Use of endostatin peptides for the treatment of fibrosis

ABSTRACT

C-terminal endostatin polypeptides are disclosed herein. In some embodiments, these polypeptides include or consist of (1) at least at least 40 consecutive amino acids of the amino acid sequence set forth as amino acids 133-180 of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4; (2) at least 40 consecutive amino acids of the amino acid sequence set forth as amino acids 133-180 of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4 with at most 5 amino acid substitutions, (3) the amino acid sequence set forth as amino acids 133-180 of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4; or (4) the amino acid sequence set forth as amino acids 133-180 of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4 with at most 5 amino acid substitutions; wherein the polypeptide has anti-fibrotic activity and wherein the polypeptide does not comprise amino acids 1-92 of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4. Polynucleotides encoding these polypeptide, host cells transformed with the polynucleotides, and methods of using these polypeptides and polynucleotides are disclosed. Uses of these polypeptide, polynucleotides and expression vectors include the treatment of fibrosis in a subject. Thus, methods are provided for treating fibrosis, including fibrosis of the skin and/or the lung.

PRIORITY CLAIM

This claims the benefit of U.S. Provisional Application No. 61/261,280,filed Nov. 13, 2009 and U.S. Provisional Application No. 61/254,143,filed Oct. 22, 2009. The prior applications are incorporated byreference herein in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support pursuantto grant AR050840 from the National Institutes of Health; the UnitedStates government has certain rights in the invention.

FIELD

This relates to the field of fibrosis, specifically to the use ofC-terminal polypeptides of endostatin for the treatment of fibrosis.

BACKGROUND

Endostatin, a 183 amino acid proteolytic cleavage fragment correspondingto the C-terminus of collagen 18, has anti-tumor activity with no toxicside effects (O'Reilly et al. (1997) Cell, 88: 277-285; Kisker et al.(2001) Cancer Res, 61:7669-7674; Dhanabal et al. (1999) Cancer Res, 59:189-197; Yoon et al. (1999) Cancer Res, 59: 6251-6256; Folkman andKalluri, (2003) Cancer Medicine, 6th edition, pp. 161-194. Hamilton: B.C. Decker Inc.). A number of anti-angiogenic activities have beenreported for this protein, such as inhibition of endothelial cellproliferation, migration, and tube formation. This activity has beenlocalized to the N-terminal region of endostatin. Endostatin alsosuppresses vascular endothelial growth factor (VEGF)-induced vascularpermeability (Takahashi et al. (2003) Faseb J, 17: 896-898). Endostatininhibits endothelial cell migration by inhibiting phosphorylation offocal adhesion kinase via binding to α5β1 integrin (Wickstrom et al.(2002) Cancer Res, 62: 5580-5589). It also has been shown that cellsurface glypicans are low-affinity endostatin receptors (Karumanchi etal. (2001) Mol Cell, 7: 811-822). Endostatin has been implicated inseveral signaling pathways, such as downregulation of c-myc (Shichiriand Hirata (2001) Faseb J, 15: 1044-1053), cyclin-D1 (Hanai et al.(2002) J Biol Chem, 277. 16464-16469) and RhoA activity (Wickstrom etal. (2003) J Biol Chem, 278: 37895-37901), blockage of VEGF signaling(Hajitou et al. (2002) Faseb J, 16: 1802-1804; Kim et al. (2002) J BiolChem, 277: 27872-27879), and inhibition of the wnt-signaling pathway(Hanai et al. (2002) J Cell Biol, 158: 529-539). Furthermore, endostatinhas been shown to bind and inactivate metalloproteinases (Kim et al.(2000) Cancer Res, 60: 5410-5413; Nyberg et al. (2003) J Biol Chem, 278:22404-22411; Lee et al. (2002) FEBS Lett, 519: 147-152) and to regulatea spectrum of genes which suppress angiogenesis (Abdollahi et al. (2004)Mol Cell, 13: 649-663).

The crystal structures of both murine and human endostatin have beenresolved (Hohenester et al. (1998) Embo J, 17: 1656-1664; Ding et al.(1998) Proc Natl Acad Sci USA, 95: 10443-10448) and show a noncovalentlyheld dimer at high concentration required for crystallization (Ding etal. (1998) Proc Natl Acad Sci USA, 95: 10443-10448). The presence of twodisulfide bonds results in a highly folded structure. Endostatin bindsone atom of zinc per monomer via the three histidines in the N-terminusof the molecule (histidines 1, 3, and 11) and asparatic 76. The heparinbinding property of endostatin is mediated by noncontiguous argininesclustered over the three dimensional globular surface of the molecule(Sasaki et al. (1999) Embo J, 18: 6240-6248).

Excessive deposition of extra cellular matrix (ECM) components such asfibronectin (FN) and type I collagen (Col1α1) by organ fibroblasts isdefined as fibrosis. Organ fibrosis is the final common pathway for manydiseases that result in end-stage organ failure. However, effectivetherapy for organ fibrosis is still unavailable (see, for example,Bjoraker et al., Am. J. Respir. Crit. Care. Med 2000; 157:199-203).Uncontrollable wound-healing responses, including acute and chronicinflammation, angiogenesis, activation of resident cells, and ECMremodeling, are thought to be involved in the pathogenesis of fibrosis(Wynn, J Clin Invest 2007; 117:524-29; Kalluri et al., Curr Opin NephrolHypertens 2000; 9:413-8). TGF-β is the prototype fibrotic cytokine thatis increased in fibrotic organs and contributes to the development offibrosis by stimulating the synthesis of ECM molecules, activatingfibroblasts to α-smooth muscle actin (α-SMA)-expressing myofibroblasts,and downregulating matrix metalloproteinases (MMPs) (see, for example,Branton et al., Microbes Infect 1999; 1:1349-65). Despite highexpectations, a clinical trial of a monoclonal anti-TGF-β antibody inpatients with early SSc failed to show any efficacy (Varga et al.,Nature Reviews Rheumatology 2009; 5:200-6). Thus, a need remains forother treatments of fibrosis.

SUMMARY

C-terminal endostatin polypeptides are disclosed herein that haveanti-fibrotic activity. In some embodiments, these polypeptides include,consist essentially of or consist of (1) at least 40 consecutive aminoacids of the amino acid sequence set forth as amino acids 133-180 of SEQID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4; (2) at least 40 consecutiveamino acids of the amino acid sequence set forth as amino acids 133-180of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4, with at most 5 aminoacid substitutions, (3) the amino acid sequence set forth as amino acids133-180 of SEQ ID NO: 2, SEQ ID NO: 13 or SEQ ID NO: 4; or (4) the aminoacid sequence set forth as amino acids 133-180 of SEQ ID NO: 2, SEQ IDNO: 13 or SEQ ID NO: 4 with at most 5 amino acid substitutions. Thesepolypeptides have anti-fibrotic activity and do not include amino acids1-92 of SEQ ID NO: 2, SEQ ID NO: 13, or SEQ ID NO: 4, respectively.Polynucleotides encoding these polypeptides, host cells transformed withthe polynucleotides, and methods of using these polypeptides andpolynucleotides are disclosed. In one example, the polypeptide includesa modification of the carboxy terminal polypeptide to include an amide.

In some embodiments, methods are disclosed for inhibiting fibrosis invivo or in vitro. In additional embodiments, methods are disclosed forthe treatment of fibrosis in a subject. In some specific non-limitingexamples, the subject has scleroderma or pulmonary fibrosis.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F. ECM production in recombinant endostatin- andendostatin-derived peptide-treated fibroblasts in combination with TGF-βstimulation. A: FN and Col1α1 expression in human normal lungfibroblasts (NL) treated with vehicle (V), rE alone, or both with priorTGF-β stimulation. Proteins were detected western blot. GAPDH was usedas a loading control for lysates. B: FN and Col1α1 expression ofendostatin polypeptide-treated lung fibroblasts following TGF-βstimulation in primary pulmonary fibroblasts from a healthy control, apatient with SSc, and a patient with IPF. C: Graphical summary of FN andCol1α1 expression in lung fibroblasts obtained using fibroblasts from 4healthy controls (NL), 3 patients with SSc, and 3 patients with IPF.Intensity of bands was normalized to that of GAPDH and expressed as aratio to Vehicle (V). Paired-t test was used for statistical analysis. *P<0.04, ** P<0.01. D: Representative result of FN and Col1α1 levels inhuman skin fibroblasts obtained from a patient with morphea and apatient with SSc. E: Representative result of FN and Col1α1 expressionin fibrotic fibroblasts obtained from a patients with IPF treated withV, 5 μg/ml of rE, or endostatin polypeptides alone (left). IPFfibroblasts were treated with different concentrations (5, 10, and 20μg/ml) of E4. DMSO (V) was added in a volume equivalent to that in thelane corresponding to 20 μg/ml of E4 (right). F: α-SMA levels in normallung fibroblasts treated with endostatin polypeptides following TGF-βstimulation.

FIGS. 2A-2C. Ex vivo human skin fibrosis organ culture model. A:Recombinant TGF-β or 1×PBS (vehicle) was injected intradermally intohuman skin explants at a concentration of 1, 10, 50 ng/ml. Skin washarvested 1 week post-injection. Representative H&E images are shown inthe upper row, and images of Masson trichrome-stained section are shownin the lower row. Magnification, 20×. B: Recombinant endostatin (rE) wasinjected into human skin at a concentration of 1, 5, 10 μg/ml. 1×PBS wasused as a vehicle control (V). Representative H&E images are shown.Magnification, 20×. C: Endostatin polypeptides (E-1, E-2, E-3, and E-4;all at 10 μg/ml) were injected intradermally in human skin. DMSO wasused as a vehicle control (V). Representative H&E images are shown.Magnification, 20×.

FIGS. 3A-3B. The effect of recombinant endostatin on TGF-β-inducedfibrosis and dermal thickness in human skin. A: Representative H&Eimages of human skin injected with Vehicle, 10 ng/ml TGF-β alone, or rE(1, 5, and 10 μg/ml) in combination with TGF-β (10 ng/ml). Tissues wereharvested one week post-injection. Magnification, 20×. B: Graphicalpresentation of dermal thickness. Data represent four independentexperiments in triplicate using human skin explants from four differentdonors. Mann-Whitney U test was used for statistical analysis. * P<0.04.

FIGS. 4A-4B. The effect of endostatin polypeptides TGF-β-inducedfibrosis and dermal thickness in human skin. A: Representative H&Eimages of human skin injected with Vehicle, 10 ng/ml TGF-β alone, orE-1, E-2, E-3, or E-4 (10 μg/ml) in combination with TGF-3 (10 ng/ml).Magnification, 20×. B: Graphical presentation of dermal thickness datashown in A. Data represent two independent experiments using human skinexplants from two donors, and each experiment was done in triplicate.Mann-Whitney U test was used for statistical analysis. * P<0.05, **P<0.02.

FIGS. 5A-5B. Dose response of E-1 and E-4 in TGF-β-induced fibrosis. A:Representative H&E images of human skin injected with E-1 (upper row) orE-4 (lower row) at a concentration of 1, 5, 10, and 20 μg/ml in thepresence of TGF-β (10 ng/ml). Magnification, 20×. B: Graphical analysisof dermal thickness data shown in A. DMSO was used as a vehicle control.Experiments were conducted in duplicate, and dermal thickness wasmeasured in 6 fields from each section. Mann-Whitney U test was used forstatistical analysis. * P<0.02, ** P<0.01.

FIGS. 6A-6B. The effect of endostatin polypeptides in the development offibrosis in vivo in mouse skin. A: Mice were injected intradermally withvehicle, 10 ng/ml TGF-β alone, or E-1, E-2, E-3, and E-4 (10 μg/ml) incombination with TGF-β (10 ng/ml). Skin was harvested after 1 weekpost-injection. Sections were stained with H&E. Magnification, 20×. B:Graphical summary of dermal thickness data shown in A. Data representfour independent experiments, each done in duplicate. Mann-Whitney Utest was used for statistical analysis. * P<0.04, ** P<0.01.

FIGS. 7A-7B. Capacity of endostatin polypeptide to inhibit tubularformation in Matrigel®. A, Representative images of Matrigel® culturesof HUVECs treated with vehicle, rE (50 nM), or E4 (50 nM). An equivalentamount of DMSO was used as vehicle. Magnification 40×. B, Imagequantification of the cord formation shown in A. Data shown summarizeresults of three independent experiments. * P<0.05, one-way ANOVAfollowed by Bonferroni's test.

FIGS. 8A-8B. The effect of endostatin E-4 on bleomycin induced dermalfibrosis in vivo. A: Mice were injected subcutaneously with 1×PBS asvehicle (V) or Bleomycin (Bleo; 20 μg/mouse) daily. E-4 (10 μg/ml) wasmixed with bleomycin on the first day, and daily bleomycinadministration was continued without subsequent injections of E4(Bleo+E-4). Skin was harvested after 10 days. Sections were stained withH&E. Magnification, 100×. B: Graphical summary of dermal thickness datashown in A. Data represent three independent experiments. Mann-Whitney Utest was used for statistical analysis. * P<0.001, ** P<0.00001. E4administration caused a significant attenuation of bleomycin induceddermal fibrosis even with a single administration of E4.

FIG. 9. E4 reverses TGF β-induced dermal fibrosis even if administered 3days following TGF-β. Mouse skin was treated with TGFβ day 1 and E-4L orE-1L (this is E-4 and E-1 administered after a 3 day lag betweenadministration of the fibrotic trigger and the administration of thepeptide. E-1 or E-4 was administered intraperitoneally (IP) at day 3 andharvested at day 7. E4 caused a significant decrease of TGFbeta induceddermal fibrosis on day 7. Thus E4 prevents (FIGS. 4-6) and reverses(FIG. 9) dermal fibrosis triggered by TGFβ.

FIG. 10A-10C. E4 attenuates bleomycin induced lung fibrosis in vivo. A:Sixty μg of bleomycin was administrated intratracheally in combinationwith DMSO as a vehicle (Bleo) or E-4 (Bleo+E-4; 10 μg/ml). In some mice,E-4 (10 μg/ml) was administered intratracheally (IT) three daysfollowing bleomycin treatment (Bleo+E-4L). PBS was used as a vehicle forbleomycin (V). Lungs were harvested 10 days post-treatment.Representative images stained with H&E (left panel) and Masson trichrome(right panel) are representative of 3 independent experiments.Magnification, 100×. E4 administered concomitantly with bleomycin orthree days following bleomycin caused a marked reduction in fibrosis andMasson Trichrome staining. B: Quantification of acid soluble collagenobtained from mouse lungs treated as in panel C with V. Bleo, Bleo+E-4,and Bleo+E-4L. The levels of collagen are presented as μg/mg (lung) fromthree independent experiments. Unpaired-t test was used for statisticalanalysis. * P<0.05. E4 polypeptide given 3 days after bleomycinsignificantly reduced collagen levels in mouse lungs C: Lowermagnification (2×) of mouse lung shown in FIG. 9 (BLM+E4/E4L IT day 10).For Bleo+E4L, Bleo was administered first, then there was a lag of threedays between Bleo and E4 administration).

FIG. 11. E4 attenuates bleomycin induced lung fibrosis in vivo whetheradministered intraperitoneally (IP) or intratracheally (IT). Bleomycinwas administered IT at day 1, and E4 was administered either IP or IT atday 3. Lungs were harvested at day 21. E4 caused a significantattenuation of bleomycin induced lung fibrosis on day 21 whetheradministered IP or IT. Thus E4 is effective at reducing fibrosisirrespective of the route of administration. Results are shown forvehicle alone (V), bleomycin alone (Bleo), bleomycin and E4 administeredIP, and bleomycin and E4 administered IT.

FIG. 12. E4 reduces fibrosis in vivo by reducing levels of lysyl oxidase(LOX), thus reducing crosslinking of collagen and rendering it lessstable and more susceptible to proteolytic degradation. Lung sections ofmice treated with BLM with or without E4 were used inimmunohistochemistry to detect LOX. The sections shown are control IgG,phosphate buffered saline, bleomycin and bleomycin followed by treatmentwith E4.

FIG. 13. E4 reduces fibrosis in vitro by blocking TGFβ-induced LOXproduction in primary human lung fibroblasts. Normal lung fibroblasts inpassage 4 were treated with vehicle, E4, TGFbeta, or TGFbeta followed 30min. later by E4. Media conditioned by the fibroblasts were analyzedusing Western blot analysis after 48 hour. Lane 1: Vehicle (DMSO); Lane2: E-4; Lane 3: TGF β; Lane 4: TGF β followed by E4. Similar resultswere obtained when LOX mRNA levels were examined by real-time PCR.

FIG. 14A-14B. A. E4 reduces fibrosis in vitro by inducing MMP-2 activityin primary human lung fibroblasts, thus resulting in increaseddegradation of collagen and other matrix proteins. Digital image of agelatin zymography gel showing increased MMP-2 activity when primaryhuman lung fibroblasts are treated with E-4 following TGFβ (lane 4).Lane 1: Vehicle (DMSO); Lane 2: E-4; Lane 3: TGF β; Lane 4: TGF βfollowed by E4. B. Digital image showing that both total and activeMMP-2 levels are increased in cells treated with TGFbeta and E-4. Thissuggests E-4 increases levels of MMP-2 pro-enzyme, but also increaseslevels of active matrix metalloproteinase (MMP-2, also calledmetalloprotease-2).

FIG. 15. E4 reduces fibrosis in vitro by inducing expression of ID1, aninhibitor of TGF β, in primary human lung fibroblasts. Real-time PCRanalysis was performed to determine the ID1 mRNA levels under theindicated conditions.

FIG. 16. E-4 reduces fibrosis in vitro by reducing levels of the mastertranscription factor Egr-1 in primary human lung fibroblasts. Reductionof Egr-1 levels parallels that of collagen, SMA, and fibronectin. Lane1: vehicle (DMSO); Lane 2: E4; Lane 3: TGFβ, Lane 4 TGFβ followed by E4after 60 minutes. The samples were harvested after 24 hours.

FIG. 17A-17B. The effect of endostatin peptides on established fibrosistriggered by TGF-b in human skin. A: Vehicle (DMSO), E-1, or E-4 (10mg/ml) was additionally injected to human skin 2 dayspost-administration of 10 ng/ml TGF-b (V, E-1L and E-4L, respectively).Representative H&E images of human skin were shown. Magnification, 20×.B: Graphical presentation of dermal thickness data shown in A. Datarepresent two independent experiments using human skin explants from twodonors, and each experiment was done in duplicate. Mann-Whitney U testwas used for statistical analysis. * P<0.01.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile [Sequence_Listing.txt, Oct. 22, 2010, 12.5 kilobytes], which isincorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is an exemplary nucleic acid sequence encoding humanendostatin.

SEQ ID NO: 2 is the amino acid sequence of human endostatin.

SEQ ID NO: 3 is an exemplary nucleic acid sequence encoding mouseendostatin.

SEQ ID NO:4 is the amino acid sequence of mouse endostatin.

SEQ ID NO: 5 is an exemplary nucleic acid sequence encoding a humanimmunoglobulin (Ig)G₁ protein.

SEQ ID NO: 6 is the amino acid sequence of a human IgG₁ protein.

SEQ ID NO: 7 is an exemplary nucleic acid sequence encoding a linker.

SEQ ID NO: 8 is an amino acid sequence of a linker.

SEQ ID NO: 9 is a portion of the rat endostatin polypeptide.

SEQ ID NO: 10 is a portion of the cow endostatin polypeptide.

SEQ ID NO: 11 is a portion of the human collagen XV polypeptide.

SEQ ID NO: 12 is an exemplary nucleic acid sequence encoding anendostatin.

SEQ ID NO: 13 is an amino acid sequence of an exemplary amino acidsequence of endostatin that differs from SEQ ID NO: 2 by three aminoacid substitutions.

DETAILED DESCRIPTION

C-terminal endostatin polypeptides are disclosed herein. In someembodiments, these polypeptides include, consist essentially of, orconsist of (1) at least 40 consecutive amino acids of the amino acidsequence set forth as amino acids 133-180 of SEQ ID NO: 13 or SEQ ID NO:2; (2) at least 40 consecutive amino acids of the amino acid sequenceset forth as amino acids 133-180 of SEQ ID NO: 13 or SEQ ID NO: 2, withat most 5 amino acid substitutions, (3) the amino acid sequence setforth as amino acids 133-180 of SEQ ID NO: 13 or SEQ ID NO: 2; or (4)the amino acid sequence set forth as amino acids 133-180 of SEQ ID NO:13 or SEQ ID NO: 2 with at most 5 amino acid substitutions; wherein thepolypeptide has anti-fibrotic activity and wherein the polypeptide doesnot comprise amino acids 1-92 of SEQ ID NO: 13 or SEQ ID NO: 2. In someembodiments, the polypeptide is amidated at the C-terminus.Polynucleotides encoding these polypeptides, host cells transformed withthe polynucleotides, and methods of using these polypeptides andpolynucleotides are disclosed. These methods include the treatment offibrosis in a subject. For example, methods are provided for treatingfibrotic conditions of the lung and the skin. In some embodiments, theanti-fibrotic C-terminal endostatin polypeptides disclosed herein canselectively inhibiting fibrosis. In some examples, fibrosis is inhibitedwithout inhibiting angiogenesis. Thus, the C-terminal endostatinpolypeptides can be used to more specifically and selectively targetunwanted fibrosis, without interfering with angiogenesis, to achieve adesired therapeutic outcome.

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Amidation or amide derivative: A post-translational modification to forman amide that can enhance the biological activity of the polypeptide. Inamidation, the C-terminal amino acid (polypeptide-COOH) is modified toform and amide (polypeptide-CONH₂). The amide may be formed bypost-translational C-terminal amidation. The amino acid to be modifiedcan be followed by a glycine, which provides the amide group. Theprocess of post-translational amidation of a polypeptide derived from aprecursor proprotein is well characterized and involves three enzymaticsteps (Cuttitta, The Anatomical Record, 236:87-93, 1993). Step oneinvolves endoproteolytic cleavage at a pair of basic amino acids nearthe carboxy terminus of the protein. Step two involvescarboxypeptidase-mediated removal of basic residues. Step three is theamidation reaction, which involves oxidation of a terminal glycine toform the amide of the neighboring carboxy terminal amino acid. Glycineis the only known amino acid to function as an amide donor for itsneighboring amino acid. Although the free acid and amidated forms of apolypeptide are difficult to distinguish structurally, the amide can be100-1000 times more biologically active than the free acid form of thepolypeptide (Cuttitta, The Anatomical Record, 236:87-93, 1993).C-terminal amidation is essential to the biological activity of manypolypeptides

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Collagen: Proteins that are found in the form of elongated fibrils inmammals that are mostly found in fibrous tissues such as tendon,ligament and skin, and is also abundant in cornea, cartilage, bone,blood vessels, the gut, and intervertebral disc. The tropocollagen or“collagen molecule” is a subunit of larger collagen aggregates such asfibrils. It is approximately 300 nm long and 1.5 nm in diameter, made upof three polypeptide strands (called alpha chains), each possessing theconformation of a left-handed helix. In type I collagen, eachtriple-helix associates into a right-handed super-super-coil that isreferred to as the collagen microfibril. Endostain is the first 183amino acids of collagen.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor of a C-terminal endostain polypeptide, such as the ability of thepolypeptide to inhibit fibrosis. Specific, non-limiting examples of aconservative substitution include the following examples:

Original Residue Conservative Substitutions Al Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Non-conservative substitutions are thosethat reduce an activity, such as the ability of a protein to inhibitfibrosis.

Consists Essentially Of/Consists Of: With regard to a polypeptide, apolypeptide that consists essentially of a specified amino acid sequenceif it does not include any additional amino acid residues. However, thepolypeptide can include additional non-peptide components, such aslabels (for example, fluorescent, radioactive, or solid particlelabels), sugars or lipids. With regard to a polypeptide, a polypeptidethat consists of a specified amino acid sequence does not include anyadditional amino acid residues, nor does it include additionalbiological components, such as nucleic acids lipids, sugars, nor does itinclude labels. A polypeptide that consists or consists essential of aspecified amino acid sequence can be glycosylated or have an amidemodification. With regard to a polynucleotide, a polynucleotide thatconsists essentially of a specified nucleic acid sequence if it does notinclude any additional nucleic acid residues. However, the polypeptidecan include additional non-nucleic acid components, such as labels (forexample, fluorescent, radioactive, or solid particle labels) orpolypeptides. With regard to a polynucleotide, a polynucleotide thatconsists of a specified nucleic acid sequence does not include anyadditional nucleic acid residues, nor does it include additionalbiological components, such as proteins, nor does it include labels.

Degenerate variant: A polynucleotide encoding a C-terminal endostatinpolypeptide that includes a sequence that is degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in this disclosure as long as the amino acidsequence of the C-terminal endostatin polypeptide encoded by thenucleotide sequence is unchanged.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (such as themetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques can also be used to provide fortranscription of the nucleic acid sequences.

Endostatin: A 183 amino acid proteolytic cleavage fragment correspondingto the C-terminus of collagen XVIII. C-terminal polypeptides ofendostatin include consecutive amino acids from the C-terminal region,which is from amino acid 93 to amino acid 183. Exemplary humanendostatin polypeptides are set forth in SEQ ID NO: 2 and SEQ ID NO: 13.

Fibrosis: The formation or development of excess fibrous connectivetissue in an organ or tissue as a reparative or reactive process, asopposed to a formation of fibrous tissue as a normal constituent of anorgan or tissue. Skin and lungs are susceptible to fibrosis. Exemplaryfibrotic conditions are scleroderma idiopathic pulmonary fibrosis,morphea, fibrosis as a result of Graft-Versus-Host Disease (GVHD),keloid and hypertrophic scar, and subepithelial fibrosis, endomyocardialfibrosis, uterine fibrosis, myelofibrosis, retroperitoneal fibrosis,nephrogenic systemic fibrosis, scarring after surgery, asthma,cirrhosis/liver fibrosis, aberrant wound healing, glomerulonephritis,and multifocal fibrosclerosis.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to endostatin originates from a nucleicacid that does not encode endostatin. In specific, non-limitingexamples, a polypeptide comprising an C-terminal endostatin polypeptideand a heterologous amino acid sequence includes an Ig (such as IgG₁)β-galactosidase, a maltose binding protein, and albumin, hepatitis Bsurface antigen, or an immunoglobulin amino acid sequence. Generally, anantibody that specifically binds to a protein of interest such asendostatin will not specifically bind to a heterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Idiopathic Pulmonary Fibrosis: A condition also known as cryptogenicfibrosing alveolitis (CFA) that is a chronic, progressive form of lungdisease characterized by fibrosis of the supporting framework(interstitium) of the lungs. By definition, the term is used only whenthe cause of the pulmonary fibrosis is unknown (“idiopathic”). When lungtissue from patients with IPF is examined under a microscope by apathologist, it shows a characteristic set of histologic/pathologicfeatures known as usual interstitial pneumonia (UIP). UIP ischaracterized by progressive scarring of both lung that involves thesupporting framework (interstitium) of the lung.

Inhibiting or treating a disease: Inhibiting a disease, such asfibrosis, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms of afibrosis, such as the formation of scar tissue or an increase in rangeof motion or a decrease in pain. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition related to the disease, such as the fibrosis.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Keloid or keloidal scar: A type of scar, which depending on itsmaturity, is composed of mainly either type III (early) or type I (late)collagen. It is a result of an overgrowth of granulation tissue(collagen type 3) at the site of a healed skin injury which is thenslowly replaced by collagen type 1. Keloids are firm, rubbery lesions orshiny, fibrous nodules, and can vary from pink to flesh-colored or redto dark brown in color. A keloid scar is benign, non-contagious, andusually accompanied by severe itchiness, sharp pains, and changes intexture. In severe cases, it can affect movement of skin. Keloids aredifferent than hypertrophic scars, which are raised scars that do notgrow beyond the boundaries of the original wound.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in the between the C-terminal endostatin polypeptides disclosedherein to provide rotational freedom to the linked polypeptide domainsand thereby to promote proper domain folding and presentation to theMHC. By way of example, in a recombinant polypeptide comprising twoC-terminal endostatin polypeptides, linker sequences can be providedbetween them, such as a polypeptide comprising C-terminal endostatinpolypeptide-linker-C-terminal endostatin polypeptide. Linker sequences,which are generally between 2 and 25 amino acids in length, are wellknown in the art and include, but are not limited to, theglycine(4)-serine spacer (×3) described by Chaudhary et al., Nature339:394-397, 1989.

Lysyl oxidase (LOX): Lysyl oxidase is an extracellular copper enzymethat catalyzes formation of aldehydes from lysine residues in collagenand elastin precursors. These aldehydes are highly reactive, and undergospontaneous chemical reactions with other lysyl oxidase-derived aldehyderesidues, or with unmodified lysine residues. This results incross-linking collagen and elastin, which is essential for stabilizationof collagen fibrils and for the integrity and elasticity of matureelastin.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Matrix metalloproteinase-2: A 72 kDa type IV collagenase also known asgelatinase A. Proteins of the matrix metalloproteinase (MMP) family areinvolved in the breakdown of extracellular matrix in normalphysiological processes, such as embryonic development, reproduction,and tissue remodeling, as well as in disease processes, such asarthritis and metastasis. Most MMP's are secreted as inactiveproproteins which are activated when cleaved by extracellularproteinases. MMP-2 degrades type IV collagen, the major structuralcomponent of basement membranes. MMP-2 also degrades additionalsubstrates such as native and denatured collagen I and fibronectin (seethe clip.ubc.ca/archive/mmp_timp_folder/mmp_substrates.shtm website).

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a polypeptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence, such as a sequence that encodes a C-terminal endostatinpolypeptide. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Peptide Modifications: C-terminal endostatin polypeptides includesynthetic embodiments of polypeptides described herein. In addition,analogs (non-peptide organic molecules), derivatives (chemicallyfunctionalized polypeptide molecules obtained starting with thedisclosed polypeptide sequences) and variants (homologs) of theseproteins can be utilized in the methods described herein. Eachpolypeptide of this disclosure is comprised of a sequence of aminoacids, which may be either L- and/or D-amino acids, naturally occurringand otherwise.

Peptides can be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpolypeptides, and optionally having other desirable properties. Forexample, carboxylic acid groups of the protein, whethercarboxyl-terminal or side chain, can be provided in the form of a saltof a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ester, or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ areeach independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the polypeptide,whether amino-terminal or side chain, can be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the polypeptide side chains may be converted toC₁-C₁₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques.Phenyl and phenolic rings of the polypeptide side chains may besubstituted with one or more halogen atoms, such as fluorine, chlorine,bromine or iodine, or with C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acidsand esters thereof, or amides of such carboxylic acids. Methylene groupsof the polypeptide side chains can be extended to homologous C₂-C₄alkylenes. Thiols can be protected with any one of a number ofwell-recognized protecting groups, such as acetamide groups. Thoseskilled in the art will also recognize methods for introducing cyclicstructures into the polypeptides of this invention to select and provideconformational constraints to the structure that result in enhancedstability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe polypeptide backbone and component amino acid side chains, resultingin such peptido- and organomimetics of C-terminal endostatin polypeptidehaving measurable or enhanced ability to treat fibrosis. For computermodeling applications, a pharmacophore is an idealized three-dimensionaldefinition of the structural requirements for biological activity.Peptido- and organomimetics can be designed to fit each pharmacophorewith current computer modeling software (using computer assisted drugdesign or CADD). See Walters, “Computer-Assisted Modeling of Drugs,” inKlegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, InterpharmPress: Buffalo Grove, Ill., pp. 165-174 and Principles of Pharmacology,Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.Also included are mimetics prepared using such techniques.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

A “therapeutically effective amount” is a quantity of a composition toachieve a desired effect in a subject being treated. For instance, thiscan be the amount necessary to induce an immune response, inhibitfibrosis, reduce scar volume or to measurably alter outward symptoms ofthe fibrotic condition. When administered to a subject, a dosage willgenerally be used that will achieve target tissue concentrations (forexample, in skin cells or lung tissue) that has been shown to achieve anin vitro effect.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Peptide or Polypeptide: Any chain of amino acids, regardless of lengthor post-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is a C-terminalendostatin polypeptide. A polypeptide can be between 5 and 60 aminoacids in length. In one embodiment, a polypeptide is from about 10 toabout 55 amino acids in length. In yet another embodiment, a polypeptideis from about 20 to about 50 amino acids in length. In yet anotherembodiment, polypeptide is about 50 amino acids in length. With regardto polypeptides, the word “about” indicates integer amounts. Thus, inone example, a polypeptide “about” 50 amino acids in length is from 49to 51 amino acids in length.

Post-translational modification: The modification of a newly formedprotein; may involve deletion of amino acids, chemical modification ofcertain amino acids (for example, amidation, acetylation,phosphorylation, glycosylation, formation of pyroglutamate,oxidation/reduction of sulfa group on a methionine, or addition ofsimilar small molecules) to certain amino acids

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, of about 15 nucleotides or morein length. Primers may be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then extended along the target DNA strand bya DNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example by polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise about 20, 25, 30, 35, 40, 50 or more consecutivenucleotides.

Purified: The C-terminal endostatin polypeptides disclosed herein can bepurified (and/or synthesized) by any of the means known in the art (see,e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185,Academic Press, San Diego, 1990; and Scopes, Protein Purification:Principles and Practice, Springer Verlag, New York, 1982). Substantialpurification denotes purification from other proteins or cellularcomponents. A substantially purified protein is at least about 60%, 70%,80%, 90%, 95%, 98% or 99% pure. Thus, in one specific, non-limitingexample, a substantially purified protein is 90% free of other proteinsor cellular components.

Thus, the term purified does not require absolute purity; rather, it isintended as a relative term. For example, a purified nucleic acid is onein which the nucleic acid is more enriched than the nucleic acid in itsnatural environment within a cell. In additional embodiments, a nucleicacid or cell preparation is purified such that the nucleic acid or cellrepresents at least about 60% (such as, but not limited to, 70%, 80%,90%, 95%, 98% or 99%) of the total nucleic acid or cell content of thepreparation, respectively.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Scleroderma: A chronic autoimmune disease characterized by fibrosis (orhardening), vascular alterations, and autoantibodies. There are twomajor forms, one is a systemic form that includes limited cutaneousscleroderma mainly affects the hands, arms and face, although pulmonaryhypertension is frequent. Diffuse cutaneous scleroderma (or systemicsclerosis) is rapidly progressing and affects a large area of the skinand one or more internal organs, frequently the kidneys, esophagus,heart and lungs. Systemic scleroderma in both of its forms can be fatal.The other form of scleroderma is a localized form that has two subtypes:morphea and linear scleroderma. The disclosed endostatin peptides can beused to treat any form of scleroderma.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (for example, GC v. ATcontent), and nucleic acid type (for example, RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific example of progressively higher stringency conditions is asfollows: 2×SSC/0.1% SDS at about room temperature (hybridizationconditions); 0.2×SSC/0.1% SDS at about room temperature (low stringencyconditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringencyconditions); and 0.1×SSC at about 68° C. (high stringency conditions).One of skill in the art can readily determine variations on theseconditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a C-terminal endostatin polypeptide will possessa relatively high degree of sequence identity when aligned usingstandard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higginsand Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a C-terminal endostatin polypeptide aretypically characterized by possession of at least 75%, for example atleast 80%, sequence identity counted over the full length alignment withthe amino acid sequence of endostatin using the NCBI Blast 2.0, gappedblastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and can possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Therapeutically effective amount: A quantity of compound, such as theC-terminal endostatin polypeptide sufficient to achieve a desired effectin a subject being treated. For instance, this can be the amountnecessary to treat or ameliorate fibrosis, such as skin or lungfibrosis, in a subject. In some embodiments, it is the amount necessaryto treat a subject by a measurable amount over a period of time, or tomeasurably inhibit progression of disease, in a subject. In otherembodiments, a therapeutically effective amount is the amount necessaryto prophylactically inhibit a disease.

An effective amount of a C-terminal endostatin polypeptide may beadministered in a single dose, or in several doses, for example daily,during a course of treatment. However, the effective amount will bedependent on the compound applied, the subject being treated, theseverity and type of the affliction, and the manner of administration ofthe compound.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art. Vectors includeplasmid vectors, including plasmids for expression in gram negative andgram positive bacterial cell. Exemplary vectors include those forexpression in E. coli and Salmonella. Vectors also include viralvectors, such as, but are not limited to, retrovirus, orthopox, avipox,fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus,baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.Vectors also include vectors for expression in yeast cells and insectcells.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or peptides areapproximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

C-Terminal Endostatin Polypeptides

C-terminal endostatin polypeptides and variants thereof are disclosedherein that inhibit fibrosis, such as found in fibrotic conditions, suchas but not limited to scleroderma. The polypeptides comprise aC-terminal amino acid sequence of an endostatin protein, but do notinclude full length endostatin. The endostatin protein can be amammalian protein, such as from a human, a non-human primate, a canine,a feline, an equine, a bovine, an ovine, a sheep, or a rodent (e.g.,mouse or rat). An exemplary nucleotide sequence encoding humanendostatin (the amino acid sequence set forth as SEQ ID NO: 2) is:

(SEQ ID NO: 1)ATGCACAGCC ACCGCGACTT CCAGCCGGTG CTCCACCTGG TTGCGCTCAA CAGCCCCCTGTCAGGCGGCA TGCGGGGCAT CCGCGGGGCC GACTTCCAGT GCTTCCAGCA GGCGCGGGCCGTGGGGCTGG CGGGCACCTT CCGCGCCTTC CTGTCCTCGC GCCTGCAGGA CCTGTACAGCATCGTGCGCC GTGCCGACCG CGCAGCCGTG CCCATCGTCA ACCTCAAGGA CGAGCTGCTGTTTCCCAGCT GGGAGGCTCT GTTCTCAGGC TCTGAGGGTC CGCTGAAGCC CGGGGCACGCATCTTCTCCT TTAACGGCAA GGACGTCCTG ACCCACCCCA CCTGGCCCCA GAAGAGCGTGTGGCATGGCT CGGACCCCAA CGGGCGCAGG CTGACCGAGA GCTACTGTGA GACGTGGCGGACGGAGGCTC CCTCGGCCAC GGGCCAGGCC TACTCGCTGC TGGGGGGCAG GCTCCTGGGGCAGAGTGCCG CGAGCTGCCA TCACGCCTAC ATCGTGCTAT GCATTGAGAA CAGCTTCATGACTGCCTCCA AGTAGSee also GENBANK® Accession Nos. NM030582.3; NM130444.2; NM130445.2, allof which are incorporated herein by reference.Another exemplary nucleotide sequence encoding a human endostatin (theamino acid sequence set forth as SEQ ID NO: 13) is:

(SEQ ID NO: 12)CACAGCCACCGC GACTTCCAGC CGGTGCTCCACCTGGTTGCG CTCAACAGCC CCCTGTCAGGCGGCATGCGG GGCATCCGCG GGGCCGACTTCCAGTGCTTC CAGCAGGCGC GGGCCGTGGGGCTGGCGGGC ACCTTCCGCG CCTTCCTGTCCTCGCGCCTG CAGGACCTGT ACAGCATCGTGCGCCGTGCC GACCGCGCAG CCGTGCCCATCGTCAACCTC AAGGACGAGC TGCTGTTTCCCAGCTGGGAG GCTCTGTTCT CAGGCTCTGAGGGTCCGCTG AAGCCCGGGG CACGCATCTTCTCCTTTGAC GGCAAGGACG TCCTGAGGCACCCCACCTGG CCCCAGAAGA GCGTGTGGCATGGCTCGGAC CCCAACGGGC GCAGGCTGACCGAGAGCTAC TGTGAGACGT GGCGGACGGAGGCTCCCTCG GCCACGGGCC AGGCCTCCTCGCTGCTGGGG GGCAGGCTCC TGGGGCAGAGTGCCGCGAGC TGCCATCACG CCTACATCGTGCTCTGCATT GAGAACAGCT TCATGACTGCCTCCAAGTAG

An exemplary human endostatin protein is:

(SEQ ID NO: 2)HSHRDFQPVL HLVALNSPLS GGMRGIRGAD FQCFQQARAV GLAGTFRAFL SSRLQDLYSIVRRADRAAVP IVNLKDELLF PSWEALFSGS EGPLKPGARI FSF N GKDVL T  HPTWPQKSVWHGSDPNGRRL TESYCETWRT EAPSATGQA Y  SLLGGRLLGQ SAASCHHAYI VLCIENSFMTASKThis protein is 183 amino acids in length, and is identical to GENBANK®Accession number AAF01310 except that it is lacking the initiatormethionine of AAF01310).Another exemplary endostatin protein is:

(SEQ ID NO: 13)HSHRDFQPVL HLVALHSPLS GGMRGIRGAD FQCFQQARAV GLAGTFRAFL SSRLQDLYSIVRRADRAAVP IVNLKDELLF PSWEALFSGS EGPLKPGARI FSF D GKDVL R  HPTWPQKSVWHGSDPNGRRL TESYCETWRT EAPSATGQA S  SLLGGRLLGQ SAASCHHAYI VLCIENSFMTASKSee also GENBANK® Accession No, CAB90482, which is incorporated hereinby reference.SEQ ID NO: 2 is identical to SEQ ID NO: 13, with the exception of threeamino acid substitutions, indicated by underlining.

An exemplary nucleotide sequence encoding mouse endostatin is:

(SEQ ID NO: 3)CATACTCATC AGGACTTTCA GCCAGTGCTC CACCTGGTGG CACTGAACAC CCCCCTGTCTGGAGGCATGC GTGGTATCCG TGGAGCAGAT TTCCAGTGCT TCCAGCAAGC CCGAGCCGTGGGGCTGTCGG GCACCTTCCG GGCTTTCCTG TCCTCTAGGC TGCAGGATCT CTATAGCATCGTGCGCCGTG CTGACCGGGG GTCTGTGCCC ATCGTCAACC TGAAGGACGA GGTGCTATCTCCCAGCTGGG ACTCCCTGTT TTCTGGCTCC CAGGGTCAAC TGCAACCCGG GGCCCGCATCTTTTCTTTTG ACGGCAGAGA TGTCCTGAGA CACCCAGCCT GGCCGCAGAA GAGCGTATGGCACGGCTCGG ACCCCAGTGG GCGGAGGCTG ATGGAGAGTT ACTGTGAGAC ATGGCGAACTGAAACTACTG GGGCTACAGG TCAGGCCTCC TCCCTGCTGT CAGGCAGGCT CCTGGAACAGAAAGCTGCGA GCTGCCACAA CAGCTACATC GTCCTGTGCA TTGAGAATAG CTTCATGACCTCTTTCTCCA AA.

An exemplary mouse endostatin protein is:

(SEQ ID NO: 4)HTHQDFQPVL HLVALNTPLS GGMRGIRGAD FQCFQQARAV GLSGTFRAFL SSRLQDLYSIVRRADRGSVP IVNLKDEVLS PSWDSLFSGS QGQLQPGARI FSFDGRDVLR HPAWPQKSVWHGSDPSGRRL MESYCETWRT ETTGATGQAS SLLSGRLLEQ KAASCHNSYI VLCIENSFMT SFSK

This protein is identical to GENBANK® Accession number AAF69009. Thenucleotide and amino acid sequences of other species are also publiclyavailable.

In one embodiment, the C-terminal endostatin polypeptide comprises about10 to about 60 consecutive amino acids of the C-terminal region of anendostatin protein, but does not include a full length endostatinprotein, or the N-terminal region of an endostatin protein. The peptidecan include from about 10 to about 55 consecutive amino acids or fromabout 20 to about 54 consecutive amino acids of the C-terminal region ofan endostatin protein, such as about 53 consecutive amino acids of theC-terminal region of an endostatin protein (such as SEQ ID NO: 2). Forexample, the peptide may include about 40, about 45, about, 46, about47, about 48, about 49, about 50, about 51, about 52 or about 53consecutive amino acids of the C-terminal region on an endostatinprotein, such as amino acids 93 to 183 of endostatin, for example SEQ IDNO: 2, SEQ ID NO: 13 or SEQ ID NO: 4. In the context of an amino acid ornucleic acid sequence, “about” means within one residue (one more or oneless than the specified number).

The endostatin peptide can include 40, 45, 46, 47, 48, 49, 50, 51, 52,53 consecutive amino acids of the C-terminal region of an endostatinprotein. In some examples the peptide consists of 40, 45, 46, 47, 48,49, 50, 51, 52, 53 consecutive amino acids of the C-terminal region ofan endostatin protein, such as but not limited to SEQ ID NO: 2, SEQ IDNO: 4 or SEQ ID NO: 13. In some embodiments, the peptide includes orconsists of at least 30 amino acids of amino acids 133 to 180 ofendostatin, or a variant thereof that has anti-fibrotic activity.

The endostatin peptide may include, consist of or consist essentially ofabout amino acid 120, 125, 130, 131, 132, 133, 134 or 135 to about aminoacids 175, 180, 181, 182 or 183 of an endostatin protein, such as SEQ IDNO: 2, SEQ ID NO: 4 or SEQ ID NO: 13. In several examples, the peptideincludes, consists of or consists essentially of amino acid 120 to 183,125 to 183, 130 to 183, 131 to 183, 132 to 183, 134 to 183, 135 to 183,120 to 180, 125 to 180, 130 to 180, 131 to 180, 132 to 180, 133 to 180,134 to 180 or 135 to 180 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 13.In specific examples, the peptide includes, consists of, or consistsessentially of amino acids 133-180 of SEQ ID NO: 2, amino acids 133-180of SEQ ID NO: 4 or amino acids 133-180 of SEQ ID NO: 13. In thiscontext, “consists essentially of” means that a peptide does not includeadditional amino acid residues but can include additional components,such as a label.

Other endostatin peptide variants disclosed herein may comprise, consistof or consist essentially of an amino acid sequence that has at leastabout 70%, 80%, 90%, 95%, 98% or 99% identity or homology with aC-terminal endostatin polypeptide. C-terminal endostatin polypeptides donot include a full length endostatin protein or the N-terminal region ofan endostatin protein (such as amino acids 1-92 of SEQ ID NO: 2).

In some non-limiting examples, C-terminal endostatin polypeptides caninclude substitutions, such as conservative amino acid substitutions, ina naturally occurring C-terminal endostatin polypeptide (see SEQ ID NO:2, 4 or 13) in at most about 1, 2, 3, 4, 5 substitutions would beexpected to retain anti-fibrotic activity. The C-terminal endostatinpolypeptide can include at most 1, at most 2, at most 3 or at most 4amino acid substitutions, such as conservative amino acid substitutions.

Peptides that are similar to the sequences described above may containsubstitutions, deletions or additions. The differences are preferably inregions that are not significantly conserved among different species.Such regions can be identified by aligning the amino acid sequences ofendostatin proteins from various animal species. Thus, the endostatinpeptide can include, consist essentially of, or consist of at least 40,at least 45, at least 46, at least 47, at least 48, at least 50, atleast 51, at least 52 or all of the amino acids set forth as amino acids133-180 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 13. Alternatively,the endostatin peptide can include at most 1, 2, 3, 4 or 5 amino acidsubstitutions in one of these sequences, provided the peptide hasanti-fibrotic activity. The peptide can be 40, 45, 46, 47, 48, 49, 50,51, 52 or 53 amino acids in length. The peptide does not include theentire sequence of endostatin, or the N-terminal region, such as of SEQID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 13. In additional embodiments, thepeptide is at most 40, 45, 46, 47, 48, 49, 50, 51, 52 or 53 amino acidsin length, such as peptide that is 40, 45, 46, 47, 48, 49, 50, 51, 52 or53 amino acids in length.

In some embodiments, the peptide is modified, such as to include aC-terminal amide. Any of the C-terminal endostatin polypeptidesdisclosed herein can include a C-terminal amide.

The following is an alignment of the human (amino acid 133-180 ofendostatin), mouse, rat, cow collagen XVIII amino acid sequences, andhuman collagen XV:

(amino acids 133-141, amino acids 145-153, amino acids 155-158, 162-166,amino acids 169-180 for the human sequence above, see SEQ ID NO: 2, forthe mouse sequence above, see SEQ ID NO: 4, for the rat sequence above,see SEQ ID NO: 9, for the cow sequence above, see SEQ ID NO: 10; humancollagen XV is SEQ ID NO: 11)

In some embodiments, the highlighted amino acids above show thoseregions of SEQ ID NO: 2, 4 or 13 should be conserved to preserveanti-fibrotic activity of the polypeptide. In further embodiments, theunderlined amino acids should be retained in order to preserveanti-fibrotic activity of the polypeptide. Thus, in some embodiments,the C-terminal endostatin polypeptide comprises amino acids 133-141,145-153, 155-158, 162-166 and 169-180 of SEQ ID NO: 2, SEQ ID NO: 4 orSEQ ID NO: 13. In one example, the A at position 145 is conserved.

E4 is amino acids 133-180 of human endostatin (see amino acids 133-180of SEQ ID NO: 2) and includes a C-terminal amide. In additionalembodiments, a region that should be retained in the peptide to retainanti-fibrotic activity is one or both potential phosphorylation sites inthe first seven amino acids of E4 that are conserved: SYCE and TWR(amino acids 1-4 and 5-7 of E4, respectively, see also amino acids133-136 and 137-139 of SEQ ID NO: 2 or SEQ ID NO: 13). In severalembodiments, regions that can be retained in the peptide to retainanti-fibrotic activity are one or both potential myristilation sites:GQaySL and GQsaAS (amino acids 15-20 and 27-32 of E4, respectively, seeamino acids 147-152 and amino acids 159-164 of SEQ ID NO: 2 or SEQ IDNO: 13). Thus, in some embodiments, the C-terminal endostatinpolypeptide includes zero, or at most 1, at most 2, at most 3, at most4, or at most 5 substitutions in amino acids 133-180 of SEQ ID NO: 2,SEQ ID NO: 4 or SEQ ID NO: 13, wherein the substitutions are not inamino acids 133-141, 145-153, 155-158, 162-166 and 169-180 of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 13, and includes a C-terminal amide.

In other embodiments, amino acids can be substituted that differ betweenthe human and mouse sequences without affecting anti-fibrotic activity.In other embodiments, the bolded and italicized amino acids shown abovein the human sequence are those amino acids that can be substitutedwhile preserving anti-fibrotic activity. For example, amino acids142-144, 154, 159-161, and 181-183 of the amino acid sequence can bealtered in the C-terminal endostatin polypeptide. These amino acids canbe substituted, for example, with those found in another species, asshown above (SEQ ID NOs: 9-10). For example, the C-terminal endostatinpolypeptide can include amino acids 133-180 of SEQ ID NO: 2 or SEQ IDNO: 13, wherein amino acids 142-144, 154, and amino acids 159-161 aresubstituted. This polypeptide can include a C-terminal amide.

Other amino acids that can be substituted, inserted or deleted at theseor other locations can be identified by mutagenesis studies coupled withbiological assays. The above alignment is provided only as a guideline.

Also encompassed herein are C-terminal endostatin polypeptides that arefused to a heterologous peptide, such as a peptide that can be used fordetecting; purifying; stabilizing; or solubilizing the endostatinpolypeptide. These polypeptides do not include a full length endostationprotein or an N-terminal region of an endostatin protein. In oneexample, a C-terminal polypeptide can be linked to an immunoglobulin(Ig) constant heavy or light chain domain or portion thereof at itsN-terminus. For example, a polypeptide, such as but not limited to aminoacids 133-180 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 13 (e.g., E4)may be linked to a CH1, CH2 and/or CH3 domain of a heavy chain. If theconstant region is from a light chain, it can be from a kappa or lambdalight chain. If the constant region is from a heavy chain, it can befrom an antibody of any one of the following classes of antibodies: IgG,IgA, IgE, IgD, and IgM. IgG can be an IgG₁, IgG₂, IgG₃ or IgG₄. Theconstant domain may be an Fc fragment. The constant domain can be from amammalian antibody, such as a human antibody. Soluble receptor-IgGfusion proteins are common immunological reagents and methods for theirconstruction are known in the art (see, for example, U.S. Pat. Nos.5,225,538, 5,726,044; 5,707,632; 750,375, 5,925,351, 6,406,697 andBergers et al. Science 1999 284: 808-12). In one example, theimmunoglobulin is the constant part of the heavy chain of human IgG,particularly IgG₁, where dimerization between two heavy chains takesplace at the hinge region. It is recognized that inclusion of the CH2and CH3 domains of the Fc region as part of the fusion polypeptideincreases the in vivo circulation half-life of the polypeptidecomprising the Fc region, and that of the oligomer or dimer comprisingthe polypeptide.

An Fc portion of human IgG₁ which includes the hinge region, and domainsCH2 and CH₃ has the nucleotide sequence:

(SEQ ID NO: 5)GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAACTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTCATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAAGAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCCAAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTCCTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTCTCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGGCAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAT GAG CTG ACCAAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATCGCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCTCCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAG CTC ACC GTG GACAAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCTCTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA,which encodes a polypeptide having the amino acid sequence:

(SEQ ID NO: 6)Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu LeuLeu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro ArgGlu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His GinAsp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro AlaPro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrLeu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu ValLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr SerLys Leu Thr Val Asp Lys Ser Arg Trp Gln Gin Gly Asn Val Phe Ser Cys Ser ValMet His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro GlyLys.Constant Ig domains can also contain one or more mutations that reduceor eliminate one or more effector function, e.g., binding to Fcreceptors and complement activation (see, for example, Morrison, Annu.Rev. Immunol., 10, pp. 239-65 (1992); Duncan and Winter (1988) Nature332: 738-740; and Xu et al. (1994) J Biol. Chem. 269: 3469-3474). Forexample, mutations of amino acids corresponding to Leu 235 and Pro 331of human IgG₁ to Glu and Ser respectively, are provided. Such constructsare further described in U.S. Pat. No. 6,656,728.

The C-terminal endostatin polypeptide can also be linked to a linkersequence with a thrombin cleavage site, such as between the C-terminalendostatin polypeptide and a heterologous polypeptide. An exemplarynucleotide sequence encoding such a site has the following nucleotidesequence: 5′ TCT AGA GGT GGT CTA GTG CCG CGC GGC AGC GGT TCC CCC GGG TTGCAG 3′ (SEQ ID NO: 7), which encodes a polypeptide having the amino acidsequence: Ser Arg Gly Gly Leu Val Pro Arg Gly Ser Gly Ser Pro Gly LeuGln (SEQ ID NO: 8). A C-terminal endostatin polypeptide can also befused to a signal sequence. For example, when prepared recombinantly, anucleic acid encoding the peptide can be linked at its 5′ end to asignal sequence, such that the peptide is secreted from the cell.

Peptides can be used as a substantially pure preparation, such aswherein at least about 90% of the peptides in the preparation are thedesired peptide. Compositions comprising at least about 50%, 60%, 70%,or 80% of the desired peptide may also be used. Peptides can bedenatured or non-denatured and may be aggregated or non-aggregated as aresult thereof.

Other C-terminal endostatin polypeptides that are encompassed herein arethose that include modified amino acids. Exemplary peptides arederivative peptides that may be one modified by glycosylation,pegylation, phosphorylation or any similar process that retains at leastone biological function of the peptide from which it was derived.Peptides may also comprise one or more non-naturally occurring aminoacids. For example, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into peptides.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyricacid, 3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,beta-alanine, fluoro-amino acids, designer amino acids such asbeta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary). In other specificembodiments, branched versions of the peptides listed herein areprovided, such as by substituting one or more amino acids within thesequence with an amino acid or amino acid analog with a free side chaincapable of forming a peptide bond with one or more amino acids (and thuscapable of forming a “branch”). Cyclical peptides are also contemplated.

Also included are peptide derivatives which are differentially modifiedduring or after synthesis, such as by benzylation, glycosylation,acetylation, phosphorylation, amidation, pegylation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. In specificembodiments, the peptides are acetylated at the N-terminus and/oramidated at the C-terminus.

In one example, the peptide includes a carboxy terminal amide. Onespecific non-limiting example of this type of C-terminal endostatinpolypeptide is E4 (see, for example, amino acids 133-180 of SEQ ID NO:13), which is described in detail in the examples section below. Thispeptide, or any of the C-terminal endostatin polypeptides disclosedherein can be amidated at the C-terminus.

Also provided are derivatives of C-terminal endostatin polypeptides,such as chemically modified peptides and peptidomimetics.Peptidomimetics are compounds based on, or derived from, peptides andproteins. Peptidomimetics can be obtained by structural modification ofknown peptide sequences using unnatural amino acids, conformationalrestraints, isosteric replacement, and the like. The subjectpeptidomimetics constitute the continum of structural space betweenpeptides and non-peptide synthetic structures; peptidomimetics may beuseful, therefore, in delineating pharmacophores and in helping totranslate peptides into nonpeptide compounds with the activity of theparent peptides.

Mimetopes of the C-terminal endostatin polypeptides are included in thepresent disclosure. Such peptidomimetics can have such attributes asbeing non-hydrolyzable (e.g., increased stability against proteases orother physiological conditions which degrade the corresponding peptide),increased specificity and/or potency for stimulating celldifferentiation. For illustrative purposes, peptide analogs can begenerated using, for example, benzodiazepines (e.g., see Freidinger etal. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988), substituted gama lactam rings(Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988, p 123), C-7 mimics (Huffmanet al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOMPublisher: Leiden, Netherlands, 1988, p. 105), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1: 1231), β-aminoalcohols (Gordonet al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986)Biochem Biophys Res Commun 134:71), diaminoketones (Nataraj an et al.(1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modified(Roark et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988, p 134). Also, see generally,Session III: Analytic and synthetic methods, in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988).

In addition to a variety of side-chain replacements which can be carriedout to generate peptidomimetics, the present disclosure specificallycontemplates the use of conformationally restrained mimics of peptidesecondary structure. Numerous surrogates have been developed for theamide bond of peptides. Frequently exploited surrogates for the amidebond include the following groups (i) trans-olefins, (ii) fluoroalkene,(iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.Additionally, peptidomimietics based on more substantial modificationsof the backbone of a peptide can be used. Peptidomimetics which fall inthis category include (i) retro-inverso analogs, and (ii) N-alkylglycine analogs (so-called peptoids). Furthermore, the methods ofcombinatorial chemistry can be used to produce peptidomimetics. Forexample, one embodiment of a so-called “peptide morphing” strategyfocuses on the random generation of a library of peptide analogs thatcomprise a wide range of peptide bond substitutes. In an exemplaryembodiment, the peptidomimetic can be derived as a retro-inverso analogof the peptide. Such retro-inverso analogs can be made according to themethods known in the art, such as that described by the Sisto et al.U.S. Pat. No. 4,522,752. A retro-inverso analog can be generated asdescribed, for example in PCT Publication No. WO 00/01720. A mixedpeptide, such as one including some normal peptide linkages, can begenerated. As a general guide, sites which are most susceptible toproteolysis are typically altered, with less susceptible amide linkagesbeing optional for mimetic switching. The final product, orintermediates thereof, can be purified by HPLC.

In some embodiments, peptides can include at least one amino acid orevery amino acid that is a D stereoisomer. Other peptides can include atleast one amino acid that is reversed. The amino acid that is reversedmay be a D stereoisomer. Every amino acid of a peptide may be reversedand/or every amino acid can be a D stereoisomer. In another illustrativeembodiment, a peptidomimetic can be derived as a retro-enantio analog ofa peptide. Retro-enantio analogs such as this can be synthesized withcommercially available D-amino acids (or analogs thereof) and standardsolid- or solution-phase peptide-synthesis techniques, as described, forexample in PCT Publication No. WO 00/01720. The final product can bepurified by HPLC to yield the pure retro-enantio analog. In stillanother illustrative embodiment, trans-olefin derivatives can be madefor the subject peptide. Trans-olefin analogs can be synthesizedaccording to the method of Y. K. Shue et al. (1987) Tetrahedron Letters28:3225 and as described in PCT Publication WO 00/01720. It is furtherpossible to couple pseudodipeptides synthesized by the above method toother pseudodipeptides, to make peptide analogs with several olefinicfunctionalities in place of amide functionalities. Still another classof peptidomimetic derivatives include the phosphonate derivatives. Thesynthesis of such phosphonate derivatives can be adapted from knownsynthesis schemes (see, for example, Loots et al. in Peptides: Chemistryand Biology, (Escom Science Publishers, Leiden, 1988, p. 118)); Petrilloet al. in Peptides: Structure and Function (Proceedings of the 9thAmerican Peptide Symposium, Pierce Chemical Co. Rockland, Ill., 1985).

Many other peptidomimetic structures are known in the art and can bereadily adapted for use in the subject peptidomimetics. For example, apeptidomimetic may incorporate the 1-azabicyclo[4.3.0]nonane surrogate(see Kim et al. (1997) J. Org. Chem. 62:2847), or an N-acyl piperazicacid (see Xi et al. (1998) J. Am. Chem. Soc. 120:80), or a 2-substitutedpiperazine moiety as a constrained amino acid analogue (see Williams etal. (1996) J. Med. Chem. 39:1345-1348). In still other embodiments,certain amino acid residues can be replaced with aryl and bi-arylmoieties, such as monocyclic or bicyclic aromatic or heteroaromaticnucleus, or a biaromatic, aromatic-heteroaromatic, or biheteroaromaticnucleus. The subject peptidomimetics can be optimized such as bycombinatorial synthesis techniques combined with high throughputscreening. Moreover, other examples of mimetopes include, but are notlimited to, protein-based compounds, carbohydrate-based compounds,lipid-based compounds, nucleic acid-based compounds, natural organiccompounds, synthetically derived organic compounds, anti-idiotypicantibodies and/or catalytic antibodies, or fragments thereof. A mimetopecan be obtained by, for example, screening libraries of natural andsynthetic compounds for compounds capable of inhibiting fibrosis. Amimetope can also be obtained, for example, from libraries of naturaland synthetic compounds, in particular, chemical or combinatoriallibraries (i.e., libraries of compounds that differ in sequence or sizebut that have the same building blocks). A mimetope can also be obtainedby, for example, rational drug design. In a rational drug designprocedure, the three-dimensional structure of a compound of the presentinvention can be analyzed by, for example, nuclear magnetic resonance(NMR) or x-ray crystallography. The three-dimensional structure can thenbe used to predict structures of potential mimetopes by, for example,computer modelling. The predicted mimetope structures can then beproduced by, for example, chemical synthesis, recombinant DNAtechnology, or by isolating a mimetope from a natural source (forexample, plants, animals, bacteria and fungi).

All of the C-terminal endostatin polypeptides of use in the disclosedmethods have anti-fibrotic activity. For example, they can reduce orinhibit fibrosis by a factor of at least about 50%, 60%, 70% 80%, 90%,or 2 fold, 5 fold, 10 fold, 30 fold or 100 fold, as compared to acontrol, such as in an assay described herein.

The C-terminal endostatin polypeptides (including amidated forms of thepeptides) can be readily synthesized by automated solid phase procedureswell known in the art. Techniques and procedures for solid phasesynthesis are described in Solid Phase Peptide Synthesis: A PracticalApproach, by E. Atherton and R. C. Sheppard, published by IRL, OxfordUniversity Press, 1989. Alternatively, these peptides may be prepared byway of segment condensation, as described, for example, in Liu et al.,Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc.117:1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res.45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu andTam, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tam, Proc. Natl.Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. PeptideProtein Res. 31:322-334, 1988). Other methods useful for synthesizingpeptides of the present disclosure are described in Nakagawa et al., J.Am. Chem. Soc. 107:7087-7092, 1985. Peptides of the disclosure can alsobe readily purchased from commercial suppliers of synthetic peptides.Such suppliers include, for example, Advanced ChemTech (Louisville,Ky.), Applied Biosystems (Foster City, Calif.), Anaspec (San Jose,Calif.), and Cell Essentials (Boston, Mass.).

Polynucleotides Encoding the C-Terminal Endostatin Polypeptides and HostCells

Polynucleotides encoding the C-terminal endostatin polypeptidesdisclosed herein are also provided. These polynucleotides include DNA,cDNA and RNA sequences which encode the peptide of interest. Silentmutations in the coding sequence result from the degeneracy (i.e.,redundancy) of the genetic code, whereby more than one codon can encodethe same amino acid residue. Thus, for example, leucine can be encodedby CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC,TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; asparticacid can be encoded by GAT or GAC; cysteine can be encoded by TGT orTGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can beencoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; andisoleucine can be encoded by ATT, ATC, or ATA. Tables showing thestandard genetic code can be found in various sources (see, for example,Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H. 5 Freeman and Co.,NY).

A nucleic acid encoding a C-terminal endostatin polypeptide can becloned or amplified by in vitro methods, such as the polymerase chainreaction (PCR), the ligase chain reaction (LCR), the transcription-basedamplification system (TAS), the self-sustained sequence replicationsystem (3SR) and the Qβ replicase amplification system (QB). Forexample, a polynucleotide encoding the protein can be isolated bypolymerase chain reaction of cDNA using primers based on the DNAsequence of the molecule. A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. PCR methods are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; andErlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotidesalso can be isolated by screening genomic or cDNA libraries with probesselected from the sequences of the desired polynucleotide understringent hybridization conditions.

The polynucleotides encoding a C-terminal endostatin polypeptide includea recombinant DNA which is incorporated into a vector in an autonomouslyreplicating plasmid or virus or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (such as a cDNA)independent of other sequences. The nucleotides can be ribonucleotides,deoxyribonucleotides, or modified forms of either nucleotide. The termincludes single and double forms of DNA.

In one embodiment, vectors are used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Several promoters are known to be ofuse in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, many inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (AMP) for propagation inbacteria. Plasmids for expression on K. lactis are known, such aspKLAC1. Thus, in one example, after amplification in bacteria, plasmidscan be introduced into the corresponding yeast auxotrophs by methodssimilar to bacterial transformation. The polynucleotides can also bedesigned to express in insect cells.

The C-terminal endostatin polypeptides can be expressed in a variety ofyeast strains. For example, seven pleiotropic drug-resistanttransporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15, togetherwith their activating transcription factors, PDR1 and PDR3, have beensimultaneously deleted in yeast host cells, rendering the resultantstrain sensitive to drugs. Yeast strains with altered lipid compositionof the plasma membrane, such as the erg6 mutant defective in ergosterolbiosynthesis, can also be utilized. Proteins that are highly sensitiveto proteolysis can be expressed in a yeast lacking the master vacuolarendopeptidase Pep4, which controls the activation of other vacuolarhydrolases. Heterologous expression in strains carryingtemperature-sensitive (ts) alleles of genes can be employed if thecorresponding null mutant is inviable.

Viral vectors can also be prepared encoding the C-terminal endostatinpolypeptides disclosed herein. A number of viral vectors have beenconstructed, including polyoma, SV40 (Madzak et al., 1992, J. Gen.Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol.Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629;Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992,Proc. Nad. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell,68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian(Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouploset al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top.Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol.,5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann etal., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990,J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol.,66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.).

Thus, in one embodiment, the polynucleotide encoding a C-terminalendostatin polypeptide is included in a viral vector. Suitable vectorsinclude retrovirus vectors, orthopox vectors, avipox vectors, fowlpoxvectors, capripox vectors, suipox vectors, adenoviral vectors, herpesvirus vectors, alpha virus vectors, baculovirus vectors, Sindbis virusvectors, vaccinia virus vectors and poliovirus vectors. Specificexemplary vectors are poxvirus vectors such as vaccinia virus, fowlpoxvirus and a highly attenuated vaccinia virus (MVA), adenovirus,baculovirus and the like.

Pox viruses of use include orthopox, suipox, avipox, and capripox virus.Orthopox include vaccinia, ectromelia, and raccoon pox. One example ofan orthopox of use is vaccinia. Avipox includes fowlpox, canary pox andpigeon pox. Capripox include goatpox and sheeppox. In one example, thesuipox is swinepox. Examples of pox viral vectors for expression asdescribed for example, in U.S. Pat. No. 6,165,460, which is incorporatedherein by reference. Other viral vectors that can be used include otherDNA viruses such as herpes virus and adenoviruses, and RNA viruses suchas retroviruses and polio.

Suitable vectors are disclosed, for example, in U.S. Pat. No. 6,998,252,which is incorporated herein by reference. In one example, a recombinantpoxvirus, such as a recombinant vaccinia virus is synthetically modifiedby insertion of a chimeric gene containing vaccinia regulatory sequencesor DNA sequences functionally equivalent thereto flanking DNA sequenceswhich in nature are not contiguous with the flanking vaccinia regulatoryDNA sequences that encode a C-terminal endostatin polypeptide. Therecombinant virus containing such a chimeric gene is effective atexpressing the C-terminal endostatin polypeptide. In one example, thevaccine viral vector comprises (A) a segment comprised of (i) a firstDNA sequence encoding a C-terminal endostatin polypeptide and (ii) apoxvirus promoter, wherein the poxvirus promoter is adjacent to andexerts transcriptional control over the DNA sequence encoding aC-terminal endostatin polypeptide; and, flanking said segment, (B) DNAfrom a nonessential region of a poxvirus genome. The viral vector canencode a selectable marker. In one example, the poxvirus includes, forexample, a thymidine kinase gene (see U.S. Pat. No. 6,998,252, which isincorporated herein by reference).

Poxviral vectors that encode a C-terminal endostatin polypeptide includeat least one expression control element operationally linked to thenucleic acid sequence encoding the C-terminal endostatin polypeptide.The expression control elements are inserted in the poxviral vector tocontrol and regulate the expression of the nucleic acid sequence.Examples of expression control elements of use in these vectorsincludes, but is not limited to, lac system, operator and promoterregions of phage lambda, yeast promoters and promoters derived frompolyoma, adenovirus, retrovirus or SV40. Additional operational elementsinclude, but are not limited to, leader sequence, termination codons,polyadenylation signals and any other sequences necessary for theappropriate transcription and subsequent translation of the nucleic acidsequence encoding the C-terminal endostatin polypeptide in the hostsystem. The expression vector can contain additional elements necessaryfor the transfer and subsequent replication of the expression vectorcontaining the nucleic acid sequence in the host system. Examples ofsuch elements include, but are not limited to, origins of replicationand selectable markers. It will further be understood by one skilled inthe art that such vectors are easily constructed using conventionalmethods (Ausubel et al., (1987) in “Current Protocols in MolecularBiology,” John Wiley and Sons, New York, N.Y.) and are commerciallyavailable.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the C-terminal endostatinpolypeptide, are known in the art. Such techniques involve, for example,homologous recombination between the viral DNA sequences flanking theDNA sequence in a donor plasmid and homologous sequences present in theparental virus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA79:7415-7419). In particular, recombinant viral vectors such as apoxviral vector can be used in delivering the gene. The vector can beconstructed for example by steps known in the art, such as stepsanalogous to the methods for creating synthetic recombinants of thefowlpox virus described in U.S. Pat. No. 5,093,258, incorporated hereinby reference. Other techniques include using a unique restrictionendonuclease site that is naturally present or artificially inserted inthe parental viral vector to insert the heterologous DNA.

DNA sequences encoding a C-terminal endostatin polypeptide can beexpressed in vitro by DNA transfer into a suitable host cell. The cellmay be prokaryotic or eukaryotic. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. Methods of stable transfer, meaning that the foreignDNA is continuously maintained in the host, are known in the art.

As noted above, a polynucleotide sequence encoding a C-terminalendostatin polypeptide can be operatively linked to expression controlsequences. An expression control sequence operatively linked to a codingsequence is ligated such that expression of the coding sequence isachieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto, appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

Hosts cells can include microbial, yeast, insect and mammalian hostcells.

Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, Jakoby and Pastan (eds),1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press,Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly usedmammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38,BHK, and COS cell lines, although cell lines may be used, such as cellsdesigned to provide higher expression desirable glycosylation patterns,or other features. As discussed above, techniques for the transformationof yeast cells, such as polyethylene glycol transformation, protoplasttransformation and gene guns are also known in the art (see Gietz andWoods Methods in Enzymology 350: 87-96, 2002).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a C-terminalendostatin polypeptide, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Therapeutic Methods and Pharmaceutical Compositions

The C-terminal endostatin polypeptides disclosed herein, or nucleicacids encoding the C-terminal endostatin polypeptides, can be used totreat fibrosis. In several examples, the C-terminal endostatinpolypeptides, or nucleic acid encoding these polypeptides are of use todecrease fibrosis, such as in a subject. Thus, in several embodiments,the methods include administering to a subject a therapeuticallyeffective amount of one or more of the C-terminal endostatinpolypeptides disclosed herein, or polynucleotides encoding thesepolypeptides, in order to decrease fibrosis. In some examples, theC-terminal endostatin polypeptide comprises or consists of amino acids133-180 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 13. However, any ofthe C-terminal endostatin polypeptides disclosed herein can be used todecrease fibrosis. In some embodiments, the peptides can be administeredas a unit dose.

Suitable subjects include those with a fibrosis of the skin or lungs,but fibrosis of any tissue can be treated using the methods disclosedherein. In one example, the subject has scleroderma. In other examples,the subject has idiopathic pulmonary fibrosis, morphea, fibrosis as aresult of Graft-Versus-Host Disease (GVHD), a keloid or hypertrophicscar, subepithelial fibrosis, endomyocardial fibrosis, uterine fibrosis,myelofibrosis, retroperitoneal fibrosis, nephrogenic systemic fibrosis,scarring after surgery, asthma, cirrhosis/liver fibrosis, aberrant woundhealing, glomerulonephritis, and multifocal fibrosclerosis.

In further examples, the methods are used to treat the systemic form ofscleroderma, such as limited cutaneous scleroderma or diffuse cutaneousscleroderma (or systemic sclerosis). The methods can be used to treatthe localized form of scleroderma, including morphea and linearscleroderma.

The methods can include selecting a subject in need of treatment, suchas a subject with a fibrotic disease, such as scleroderma, idiopathicpulmonary fibrosis, morphea, a keloid scar, a hypertrophic scar, orsubepithelial fibrosis. In exemplary applications, compositions areadministered to a subject having a fibrotic disease, such asscleroderma, idiopathic pulmonary fibrosis, morphea, a keloid scar, ahypertrophic scar, or subepithelial fibrosis, or any of the disorderslisted above, in an amount sufficient to reduce the fibrosis. Amountseffective for this use will depend upon the severity of the disease, thegeneral state of the patient's health, and the robustness of thepatient's immune system. In one example, a therapeutically effectiveamount of the compound is that which provides either subjective reliefof a symptom(s) or an objectively identifiable improvement as noted bythe clinician or other qualified observer.

A method is provided herein for decreasing skin thickness. The methodincludes administering a therapeutically effective amount of aC-terminal endostatin polypeptide, thereby decreasing skin thickness. Inanother embodiment, an methods is provided for decreasing lung fibrosis.The method includes administering a therapeutically effective amount ofa C-terminal endostatin polypeptide, thereby decreasing skin thickness.Any of the C-terminal endostatin polypeptides disclosed herein can beused in these methods. In some embodiments, the C-terminal endostatinpolypeptide comprises, or consists of, amino acids 133-180 of SEQ ID NO:2, SEQ ID NO: 4 or SEQ ID NO: 13.

Methods are provided herein for decreasing lysyl oxidase (LOX), such astransforming growth factor (TGF)-β induced LOX. The method includescontacting a cell with an effective amount of a C-terminal endostatinpolypeptide, thereby decreasing LOX. The methods can be practiced invivo or in vitro. In some embodiments, the methods include comparing theamount of LOX produced by a cell contacted with a C-terminal endostatinpolypeptide to a control. The control can be a standard value, or theamount of LOX produced by a cell not contacted with the C-terminalendostatin polypeptide, such as a cell contacted with a carrier.

Methods are provided herein for increasing matrix metalloproteinase-2(MMP-2). The method includes contacting a cell with an effective amountof a C-terminal endostatin polypeptide, thereby increasing MMP-2production. The methods can be practiced in vivo or in vitro. In someembodiments, the methods include comparing the amount of MMP-2 producedby a cell contacted with a C-terminal endostatin polypeptide to acontrol The control can be a standard value, or the amount of MMP-2produced by a cell not contacted with the C-terminal endostatinpolypeptide, such as a cell contacted with a carrier.

A C-terminal endostatin polypeptide can be administered by any meansknown to one of skill in the art (see Banga, A., “Parenteral ControlledDelivery of Therapeutic Peptides and Proteins,” in Therapeutic Peptidesand Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995)either locally or systemically, such as by intradermal, intrathecal,intramuscular, subcutaneous, intraperitoneal or intravenous injection,but even oral, nasal, transdermal or anal administration iscontemplated. In one embodiment, administration is by subcutaneous,intradermal, or intramuscular injection. In another embodiment,administration is by intraperitoneal or intrathecal administration. Toextend the time during which the peptide or protein is available tostimulate a response, the peptide or protein can be provided as animplant, an oily injection, or as a particulate system. The particulatesystem can be a microparticle, a microcapsule, a microsphere, ananocapsule, or similar particle. (see, e.g., Banga, supra).

For treatment of the skin, a therapeutically effective amount of atleast one C-terminal endostatin polypeptide, or a nucleic acid encodingthe peptide, can be locally administered to the affected area of theskin, such as in the form of an ointment. In one embodiment, theointment is an entirely homogenous semi-solid external agent with afirmness appropriate for easy application to the skin. Such an ointmentcan include fats, fatty oils, lanoline, Vaseline, paraffin, wax, hardointments, resins, plastics, glycols, higher alcohols, glycerol, wateror emulsifier and a suspending agent. Using these ingredients as a base,a decoy compound can be evenly mixed. Depending on the base, the mixturecan be in the form of an oleaginous ointment, an emulsified ointment, ora water-soluble ointment oleaginous ointments use bases such as plantand animal oils and fats, wax, Vaseline and liquid paraffin. Emulsifiedointments are comprised of an oleaginous substance and water, emulsifiedwith an emulsifier. They can take either an oil-in-water form (O/W) or awater-in-oil-form (W/O). The oil-in-water form (O/W) can be ahydrophilic ointment. The water-in-oil form (W/O) initially lacks anaqueous phase and can include hydrophilic Vaseline and purifiedlanoline, or it can contain a water-absorption ointment (including anaqueous phase) and hydrated lanoline. A water-soluble ointment cancontain a completely water-soluble Macrogol base as its main ingredient.

Pharmaceutically acceptable carriers include a petroleum jelly, such asVASELINE®, wherein the petroleum jelly contains 5% stearyl alcohol, orpetroleum jelly alone, or petroleum jelly containing liquid paraffin.Such carriers enable pharmaceutical compositions to be prescribed informs appropriate for consumption, such as tablets, pills, sugar-coatedagents, capsules, liquid preparations, gels, ointments, syrups,slurries, and suspensions. When locally administered into cells in anaffected area or a tissue of interest, the at least one C-terminalendostatin polypeptide, or polynucleotide encoding the peptide can beadministered in a composition that contains a synthetic or naturalhydrophilic polymer as the carrier. Examples of such polymers includehydroxypropyl cellulose and polyethylene glycol. One or more C-terminalendostatin polypeptides, or polynucleotide encoding the polypeptides,can be mixed with a hydrophilic polymer in an appropriate solvent. Thesolvent is then removed by methods such as air-drying, and the remainderis then shaped into a desired form (for example, a sheet) and applied tothe target site. Formulations containing such hydrophilic polymers keepwell as they have a low water-content. At the time of use, they absorbwater, becoming gels that also store well. In the case of sheets, thefirmness can be adjusted by mixing a polyhydric alcohol with ahydrophilic polymer similar to those above, such as cellulose, starchand its derivatives, or synthetic polymeric compounds. Hydrophilicsheets thus formed can be used. A therapeutically effective amount ofone or more C-terminal endostatin polypeptide, or polynucleotideencoding the peptide can also be incorporated into bandages anddressings.

For administration by inhalation, the C-terminal endostatin polypeptide,or polynucleotide encoding the peptide can be conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesfor use in an inhaler or insufflator can be formulated containing apowder mix of the compound and a suitable powder base such as lactose orstarch.

In some embodiments, the C-terminal endostatin polpeptide, such as, butnot limited to E4, can be administered by inhalation. For example, theC-terminal endostatin polypeptide can be administered in an aerosolizedform, such as using a nebulizer or a metered dose inhabler. Technologiesof use include micropump nebulizers (such as the AEROGEN GO® system),jet nebulizers designed to produce large fine particle fractions (suchas the PARI LC STAR®), jet nebulizers developing less shear duringatomization (such as the HUDSON MICROMIST®), and ultrasonic nebulizers(such as the DeVilbiss ULTRA-NEB®).

The endostatin polypeptide can be dissolved in a carrier, such assaline, and atomized using the devices above. The associated aerosolscan be collected using a NEXT GENERATION IMPACTOR® (NGI) (MSP Corp.,Shoreview, Minn.), which uses a series of aerodynamic stages to separateand collect the aerosol into separate fractions based on droplet size.Since droplet size is the primary determinant of deposition location inthe lungs, this device allows us to specifically isolate the portion ofthe liquid aerosol that will deposit in the small airways and alveoli.

Aerosol particle size is often expressed in terms of mass medianaerodynamic diameter (MMAD), a parameter that is based on particle size,shape, and density. For a spherical particle, MMAD is equal to MMD(p^(1/2)), in which MMD is mass median diameter and r is the bulkdensity. For a non-spherical particle, MMAD is equal to MMD (p/x)^(1/2),in which X is the shape factor. Thus, particles with larger than unitdensity will have actual diameters smaller than their MMAD.

The site of particle deposition within the respiratory tract isdemarcated based on particle size. In one example, particles of about 1to about 500 microns are utilized, such as particles of about 25 toabout 250 microns, or about 10 to about 25 microns are utilized. Inother embodiments, particles of about 1 to 50 microns are utilized. Foruse in a metered dose inhaler, for administration to lungs particles ofless than about 10 microns, such as particles of about 2 to about 8microns, such as about 1 to about 5 microns, such as particles of 2 to 3microns, can be utilized.

A therapeutically effect amount of a C-terminal endostatin polypeptide,or polynucleotide encoding the peptide can be administered in thepharmaceutically acceptable carrier. Pharmacologically acceptablecarriers (e.g., physiologically or pharmaceutically acceptable carriers)are well known in the art, and include, but are not limited to bufferedsolutions as a physiological pH (e.g. from a pH of about 7.0 to about8.0, or at a pH of about 7.4). One specific, non-limiting example of aphysiologically compatible buffered solution is phosphate bufferedsaline. Other pharmacologically acceptable carriers include penetrants,which are particularly suitable for pharmaceutical formulations that areintended to be topically applied (for example in the application ofsurgical wounds to promote healing).

The pharmacological compositions disclosed herein facilitate the use ofat least one C-terminal endostatin polypeptide, or polynucleotideencoding the peptide, either in vivo or ex vivo, to decrease fibrosis.Such a composition can be suitable for delivery of the active ingredientto any suitable subject, and can be manufactured in a manner that isitself known, e.g., by means of conventional mixing, dissolving,granulating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmacological compositions can be formulated in aconventional manner using one or more pharmacologically (e.g.,physiologically or pharmaceutically) acceptable carriers, as well asoptional auxiliaries that facilitate processing of the active compoundsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen. Thus, forinjection, the active ingredient can be formulated in aqueous solutions.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the active ingredient can be combined withcarriers suitable for incorporation into tablets, pills, capsules,liquids, gels, syrups, slurries, suspensions and the like. The activeingredient can be formulated for parenteral administration by injection,such as by bolus injection or continuous infusion. Such compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Other pharmacological excipientsare known in the art.

Optionally, the at least one C-terminal endostatin polypeptide, orpolynucleotide encoding the peptide can be contained within orconjugated with a heterologous protein, hydrocarbon or lipid, whetherfor in vitro or in vivo administration. Co-administration can be suchthat the at least one C-terminal endostatin polypeptide, orpolynucleotide encoding the peptide is administered before, atsubstantially the same time as, or after the protein, hydrocarbon, orlipid. In one embodiment, the at least one C-terminal endostatinpolypeptide, or polynucleotide encoding the peptide is administered atsubstantially the same time, as the protein, hydrocarbon, or lipid.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compositions of the invention described above,increasing convenience to the subject and the physician. Many types ofrelease delivery systems are available and known to those of ordinaryskill in the art. They include polymer based systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems, such as lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono- di- and tri-glycerides; hydrogel release systems; silasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the at least one C-terminal endostatin polypeptide, orpolynucleotide encoding the peptide is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775; 4,667,014;4,748,034; 5,239,660; and 6,218,371 and (b) diffusional systems in whichan active component permeates at a controlled rate from a polymer suchas described in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions, such as scleroderma.Long-term release, as used herein, means that the implant is constructedand arranged to deliver therapeutic levels of the active ingredient forat least 30 days, and preferably 60 days. Long-term sustained releaseimplants are well known to those of ordinary skill in the art andinclude some of the release systems described above. These systems havebeen described for use with oligodeoxynucleotides (see U.S. Pat. No.6,218,371). For use in vivo, nucleic acids and peptides are preferablyrelatively resistant to degradation (such as via endo- andexo-nucleases). Thus, modifications, such as the inclusion of aC-terminal amide, can be used.

The therapeutically effective amount of C-terminal endostatinpolypeptide, or polynucleotide encoding the peptide will be dependent onthe C-terminal endostatin polypeptide, or polynucleotide encoding thepeptide, that is utilized, the subject being treated, the severity andtype of the affliction, and the manner of administration. For example, atherapeutically effective amount of a polynucleotide encoding thepeptide can vary from about 0.01 μg per kilogram (kg) body weight toabout 1 g per kg body weight, such as about 1 μg to about 5 mg per kgbody weight, or about 5 μg to about 1 mg per kg body weight. The exactdose is readily determined by one of skill in the art based on thepotency of the specific compound the age, weight, sex and physiologicalcondition of the subject.

With regard to the administration of nucleic acids, one approach toadministration of nucleic acids is direct treatment with plasmid DNA,such as with a mammalian expression plasmid. As described above, thenucleotide sequence encoding a N-terminal endostatin peptide can beplaced under the control of a promoter to increase expression of themolecule.

When a viral vector is utilized for administration in vivo, it isdesirable to provide the recipient with a dosage of each recombinantvirus in the composition in the range of from about 10⁵ to about 10¹⁰plaque forming units/mg mammal, although a lower or higher dose can beadministered. The composition of recombinant viral vectors can beintroduced into a mammal either prior to any evidence of a cancer, or tomediate regression of the disease in a mammal afflicted with the cancer.Examples of methods for administering the composition into mammalsinclude, but are not limited to, exposure of cells to the recombinantvirus ex vivo, or injection of the composition into the affected tissueor intravenous, subcutaneous, intradermal or intramuscularadministration of the virus. Alternatively the recombinant viral vectoror combination of recombinant viral vectors may be administered locallyby direct injection into the cancerous lesion in a pharmaceuticallyacceptable carrier. Generally, the quantity of recombinant viral vector,carrying the nucleic acid sequence of one or more C-terminal endostatinpolypeptides to be administered is based on the titer of virusparticles. An exemplary range of the immunogen to be administered is 10⁵to 10¹⁰ virus particles per mammal, such as a human.

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 10 mg ofC-terminal endostatin polypeptide per patient per day. Dosages from 0.1up to about 100 mg per patient per day can be used, particularly if theagent is administered to a secluded site and not into the circulatory orlymph system, such as into a body cavity or into a lumen of an organ.Actual methods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the subject. Systemic or local administrationcan be utilized.

In a further method, an additional agent is administered. In oneexample, this administration is sequential. In other examples, theadditional agent is administered simultaneously with the C-terminalendostatin polypeptide.

For the treatment of scleroderma, examples of additional agents that canbe used with a C-terminal endostatin polypeptides include nifedipine,amlodipine, diltiazem, felodipine, or nicardipine. An investigationaldrug Gleevec, is also used for the treatment of scleroderma. Gleevec orother tyrosine kinase inhibitors can be used with the C-terminalendostatin polypeptides disclosed herein. Patients with lung involvementof scleroderma benefit from oxygen therapy; the C-terminal endostatinpolypeptides disclosed herein can be administered with this therapy.

For the treatment of fibrosis of the skin and scleroderma, additionalagents of use are d-penicillamine, colchicine, Relaxin, steroids, andcyclosporine. C-terminal endostatin polypeptides also can be used incombination with immunosuppressive agents. Additionally, the C-terminalendostatin polypeptides can be used with methotrexate, cyclophosphamide,azathioprine, mycophenolate, glitazones, endothelin receptorantagonists, or Fulvestrant (ICI-182,780).

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Excessive deposition of extra cellular matrix (ECM) components such asfibronectin (FN) and type I collagen (Col1α1) by organ fibroblasts isdefined as fibrosis. Organ fibrosis is the final common pathway for manydiseases that result in end-stage organ failure. However, effectivetherapy for organ fibrosis is still unavailable (see, for example,Bjoraker et al., Am. J. Respir. Crit. Care. Med 2000; 157:199-20; Vargaand Abraham, J Clin Invest 2007; 117:557-67; Wynn, J Clin Invest 2007;117:524-29). Uncontrollable wound-healing responses, including acute andchronic inflammation, angiogenesis, activation of resident cells, andECM remodeling, are thought to be involved in the pathogenesis offibrosis (Wynn, J Clin Invest 2007; 117:524-29; Kalluri and Sukhatme.Curr Opin Nephrol Hypertens 2000; 9:413-8). TGF-β is the prototypefibrotic cytokine that is increased in fibrotic organs and contributesto the development of fibrosis by stimulating the synthesis of ECMmolecules, activating fibroblasts to α-smooth muscle actin(α-SMA)-expressing myofibroblasts, and downregulating matrixmetalloproteinases (MMPs) (Branton, Microbes Infect 1999; 1:1349-65;Varga and Pasche Nature Reviews Rheumatology 2009; 5:200-6). Despitehigh expectations, a clinical trial of a monoclonal anti-TGF-β antibodyin patients with early SSc failed to show any efficacy (Varga andPasche, Nature Reviews Rheumatology 2009; 5:200-6).

Endostatin is a 20-kDa internal fragment of the carboxyterminus ofcollagen XVIII. It was originally identified in the supernatant of acultured murine hemangioendothelioma cell line with potentantiangiogenic activity (O'Reilly et al., Cell 1997; 88:277-85).Endostatin inhibits endothelial proliferation and tube formation invitro, and tumor growth in vivo (Dhanabal et al., Biochem Biophys ResCommun 1999; 258:345-52). Studies have been conducted to assessendostatin's anti-tumor properties, including clinical trials (Folkman,Exp Cell Res 2006; 312:594-607). The NH₂-terminal domain of endostatinhas been reported as the functional domain responsible for inhibitingangiogenesis (Tjin Than Sjin et al., Cancer Res 2005; 65:3656-63).Although the exact molecular mechanism of its effect remains unclear,integrins, glypicans, flk-1, and nucleolin have been reported asendostatin receptors (Sudhakar et al., Proc Natl Acad Sci USA 2003;100:4766-71; Karumanchi et al., Mol Cell 2001; 7:811-22). Recent studieshave shown that endostatin is increased in serum and/or BALF obtainedfrom IPF and SSc patients with pulmonary fibrosis (for example, Sumi, JClin Lab Anal 2005; 19:146-9).

In the studies presented herein, the effects of endostatin on fibrosiswere evaluated. The effect of endostatin and endostatin-derived peptideson fibrosis in vitro was assessed using primary human fibroblasts, exvivo using human skin, and in vivo in mice skin treated with TGF-β.Surprisingly, the findings demonstrate that a carboxy-terminal peptideof endostatin has anti-fibrotic activity and provide a novel therapy forfibrotic disorders.

Example 1 Materials and Methods

Reagents and Antibodies

The full-length recombinant human endostatin (rE) was purchased fromSigma-Aldrich (St. Louis, Mo.). Recombinant human TGF-β was from R&DSystems Inc. (Minneapolis, Minn.). Mouse monoclonal anti-humanfibronectin (FN) antibody, goat polyclonal anti-human type I Collagen αIchain (Col1α1) antibody, and mouse monoclonal anti-human GAPDH antibodywere from Santa Cruz Biotechnology (Santa Cruz, Calif.). Mousemonoclonal anti-human α-smooth muscle actin (α-SMA) antibody was fromSigma-Aldrich.

Synthesis of Human Endostatin Peptides

Peptides were synthesized by the solid-phase on Liberty MicrowaveSynthesizer (CEM Corporation, 3100 Smith Farm Road, Mathews, N.C. 28106)using FMOC synthesis protocol. Briefly, synthesis was performed bystepwise addition of activated amino acids to the solid support (Wangresin and PEG-PS) starting from the carboxy terminus to the aminoterminus. Activation of amino acids was performed by DIPEA/HOBT/TBTUchemistry. At the end of the synthesis, peptides were cleaved off theresin with reagent R (90% TFA, 5% Thioanisole, 3% Ethanedithiol, and 2%Anisole) and subjected to multiple ether extractions. The crude peptideswere analyzed, characterized, and purified by Gel filtration (G-25column), Reversed-Phase High Performance Liquid Chromatography (RP-HPLC,486 and 600E by Waters Corporation). The correct mass was confirmed byMALDI-TOF Mass Spectroscopy (The Voyager-DE STR BiospectrometryWorkstation). Sequences of the peptides are shown in Table 1 andcorrespond to amino acids 1-45 (E1); 71-115 (E2); 133-180 (E3), 133-180A(E4) which differs from E3 by the presence of a carboxy-terminal amide.The purity of all peptides was >98%. All peptides were dissolved in DMSOat a concentration 5 mg/ml, and diluted in 1×PBS to 1-20 μg/ml.

Primary Fibroblast Culture

Human primary lung and skin fibroblasts were cultured. The explantedlungs of normal organ donors, patients with SSc or IPF, and clinicallyinvolved skin of SSc patients, a morphea patient and healthy donors wereused for primary fibroblast culture. Approximately 2-cm pieces ofperipheral lung and skin were minced and fibroblasts were cultured inDulbecco's modified Eagle's medium (DMEM; Mediatech, Herndon, Va.)supplemented with 10% FBS, penicillin, streptomycin, and anti-mycoticagent, as previously described (Feghali et al., Arthritis Rheum 1999;42:1451-7). All the cells were used between passages 3-6.

Western Blot Analysis

Cellular lysates were obtained from cultured fibroblasts as previouslydescribed (Pilewski et al., Am J Pathol 2005; 166:399-407). Briefly,2.0×10⁵ primary fibroblasts were cultured in 35-mm wells in 0.5%FBS-containing medium supplemented with 10 ng/ml of human recombinantTGF-β or PBS as vehicle control for 24 h, following which 5 μg/ml ofhuman rE, endostatin peptides (E1-E4), or DMSO (vehicle) was added for48 h. In some experiments, endostatin peptides were used without TGF-βstimulation. Cellular lysates were analyzed by western blot. Signalswere detected following incubation with horseradishperoxidase-conjugated secondary antibody and chemiluminescence (PerkinElmer Life Sciences, Inc., Boston, Mass.). The intensity of individualbands with expected molecular sizes was semi-quantitatively analyzedusing the image/J® software available at on the internet(/rsb.info.nih.gov/ij/index.html), and normalized to individual GAPDHintensity.

Ex Vivo Human Skin Assays

Human abdominal skin was obtained from corrective plastic surgery. Aspreviously described (Yasuoka et al., The Open Rheumatol J 2008;2:17-22), subcutaneous fat tissue was removed uniformly and skin tissuewas cut into 1.5 cm×1.5 cm sections. The following were injectedintradermally in a total volume of 100 μl 1×PBS: rE alone (1-10 μg/ml),endostatin peptides alone (10 μg/ml), rE or endostatin peptides (1-20μg/ml) in combination with TGF-β (10 ng/ml), and TGF-β alone (10 ng/ml).In some experiments, human skin was first injected with TGF-β for 48 hfollowed by recombinant endostain (rE) administration in the sameinjection site as TGF-β. Independent experiments were conducted induplicate or triplicate as indicated in the figure legends. Explantscontaining complete epidermal and dermal layers were cultured in an airliquid interface with the epidermal and keratin layers side up andexposed to air. The culture medium was replaced every other day. After 1or 2 weeks, skin tissue corresponding to an area with 8-mm diametercentered around the injection site was harvested using disposable 8-mmAcu Punch® (Acuderm inc., Lauderdale, Fla.). Skin tissue was fixed in10% formalin prior to embedding in paraffin.

In Vivo Mouse Experiments

CB57BL6/J male mice were purchased from The Jackson Laboratory (BarHarbor, Me.). Human rE (10 μg/ml) or Endostatin peptides (10 μg/ml) incombination with TGF-β (10 ng/ml), or TGF-β alone were injectedintradermally on the back of mice in a total volume of 100 μl 1×PBS.Mice were injected in two different skin sites and sacrificed one weekpost-injection. Skin surrounding the injection site was harvested andfixed in 10% formalin prior to embedding in paraffin.

Measurement of Skin Dermal Thickness

Six μm sections of paraffin-embedded human and mouse skin tissues werestained with hematoxylin and eosin (H&E). In some experiments, sectionswere stained with Masson trichrome which identifies collagens. Imageswere taken on a Nikon Eclipse 800 microscope. The thickness of thedermis was measured in 6 random fields of each section using theimage/J® software. Data are shown in arbitrary units.

Tubular Formation Assay

The ability of endostatin peptide to inhibit angiogenesis was examinedin tubular formation assay using Matrigel® culture. Human umbilical veinendothelial cells (HUVECs) were maintained in endothelial cell basalmedium-2 (EBM-2; Clonetics, San Diego, Calif.) supplemented with EBM-2MV SingleQuots®. HUVECs (5×10⁴) were cultured in duplicate on 24-wellMatrigel® plates (BD Biosciences, San Diego, Calif.) alone, or in thepresence of rE or E4 peptide (50 nM) in EBM-2 at 37° C. DMSO was used asvehicle control. After 24 hours, images were captured using a convertedmicroscope. The degree of cord formation was quantified by measuring thearea occupied by tubes in 6 random fields per well. Three independentexperiments were performed.

Statistical Analysis

All continuous variables were expressed as the mean±standard deviation.Comparisons between 2 groups were tested for statistical significanceusing the paired t-test or Mann-Whitney U test as appropriate.Comparison among 3 groups was performed using ANOVA followed byBonferroni's test.

Example 2 Human Endostatin Inhibits FN and Col1α1 Production inTGF-β-Treated Human Primary Lung and Skin Fibroblasts In Vitro

To evaluate whether endostatin modulates production of ECM components infibroblasts, FN and Col1α1 expression was examined in normal human lungfibroblasts by western blot analysis. Cells were treated with 5 μg/ml rEfor 48 h with or without pre-stimulation with human TGF-β for 24 h. Asshown FIG. 1A, rE dramatically reduced FN and Col1α1 levels in TGF-βpre-treated fibroblasts. To define the functional domain of endostatinthat mediates its inhibitory effect, four different peptides weresynthesized corresponding to different regions of endostatin (Table 1).

TABLE I Amino acid sequence of human endostatin fragments.E1 (1-45 of SEQ ID NO: 2)H-¹HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGT⁴⁵-OHE2 (71-115 of SEQ ID NO: 2)H-⁷¹IVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWP¹¹⁵-OHE3 (133-180 of SEQ ID NO: 2)H-¹³³SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT¹⁸⁰-OHE4 (133-180A of SEQ ID NO: 2)H-¹³³SYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMT¹⁸⁰-CONH2

As shown in FIGS. 1B and 1C, a fragment from the carboxy terminus ofendostatin (E4) significantly suppressed FN and Col1α1 production inTGF-β treated cells compared with normal lung fibroblasts treated withTGF-β alone (P=0.03, in both comparisons). On the other hand, E1peptide, located in the amino terminal region of endostatin, had noeffect. In addition to healthy fibroblasts, lung fibroblasts obtainedfrom SSc and IPF patients, who had clinical lung fibrosis, were used inparallel assays with similar results (FIGS. 1B and 1C). Havingdemonstrated anti-fibrotic effects of rE and E4 in lung fibroblasts, theeffects of these peptides was examined on skin fibroblasts since skin isa major organ affected by fibrosis in SSc. Primary fibroblasts obtainedfrom the skin of healthy controls, patients with systemic sclerosis(SSc) or localized scleroderma (morphea) were treated with rE or E4.Similarly to lung fibroblasts, rE and E4 reduced TGF-β-induced ECMproduction in dermal fibroblasts. Representative results are shown inFIG. 1D.

Example 3 Endostatin Peptides Reverse the Fibrotic Phenotype of PrimaryLung Fibroblasts from Patients with SSc and IPF

Since it has been shown that TGF-β is upregulated in fibrotic tissue, itwas examined if matrix production in fibrotic lung fibroblasts wasaltered by treatment with endostatin peptide in the absence of TGF-βstimulation. As shown in FIG. 1E left panel, both FN and Col1α1 levelsdecreased in E4-treated fibroblasts. In addition, the same fibroblastswere treated with different concentrations of E4 to identify the optimalanti-fibrotic dose. E4 dose-dependently reduced Col1α1 levels whencompared to vehicle control (FIG. 1E, right panel), but had a modesteffect on FN levels. The reduction in ECM was more modest than thatobserved following TGF-β stimulation. Taken together, the resultsindicate that E4 can reduce baseline production of ECM components infibroblasts from a fibrotic milieu and thus reverse the fibroticphenotype.

Myofibroblasts, activated fibroblasts which express α-SMA, are inducedby TGF-β stimulation and play a central role in fibrosis. Therefore, theeffects of endostatin peptides on α-SMA expression in normal lungfibroblasts was examined. As shown in FIG. 1F, TGF-β stimulation greatlyincreased α-SMA expression. Interestingly, E4, and to a lesser extentE3, decreased TGF-β-induced α-SMA levels suggesting that thecarboxy-terminal region of endostatin can prevent the activation offibroblasts and their transition to a myofibroblastic phenotype.

Example 4 Endostatin Reduces Dermal Thickness and Prevents TGF-β-InducedFibrosis in Human Skin

Cultured human skin explants can be used as an organ model to assess theeffects of fibrogenic factors and for evaluating the efficacy ofinhibitors/therapies to halt the progression of fibrosis and potentiallyreverse it (Yasuoka, The Open Rheumatol J 2008; 2:17-22). To evaluatethe efficacy of endostatin as a potential therapeutic agent forfibrosis, this ex vivo human skin model was used. Since TGF-β is awell-known pro-fibrotic factor that plays a central role in fibrosis,human recombinant TGF-β was first injected intradermally to assess thelevel of fibrosis. As shown in FIG. 2A, TGF-β injection dramaticallyincreased dermal thickness in a dose-dependent manner one weekpost-injection. The fibrotic effect of TGF-β (10 ng/ml) resolved by twoweeks. The baseline effects of rE (1, 5, and 10 μg/ml) or endostatinpeptides (10 μg/ml) were also examined individually. Although rE andE1-4 did not significantly alter dermal thickness, rE, E3, and E4 showeda tendency towards reduction in human dermal thickness (FIGS. 2B and2C). It was determined if rE could inhibit fibrosis in TGF-β-treatedhuman skin. TGF-β and rE were injected simultaneously. One weekpost-administration, rE in combination with TGF-β significantly reduceddermal thickness in a dose-dependent manner (FIG. 3). To assess theeffects of rE on reversing fibrosis, the peptide was injected 2 daysafter TGF-β administration. Similarly to co-treatment, delayed rE alsosignificantly ameliorated TGF-β-induced dermal fibrosis. The findingsindicate that human endostatin can prevent the development andprogression of fibrosis and also reverse TGF-β-induced fibrosis in humanskin.

Example 5 Endostatin Peptides Reduce TGF-β-Induced Fibrosis in HumanSkin Ex Vivo and Reverse Existing Fibrosis

To determine which part of endostatin is responsible for inhibitingTGF-β-induced fibrosis in human skin explants, endostatin peptides (10μg/ml) were administrated in the presence of 10 ng/ml of TGF-β.Representative images are shown in FIG. 4A. E3 and E4 significantlyabolished the development of fibrosis as measured by dermal thicknesswhen compared to TGF-β alone (P=0.04, 0.01, respectively; FIG. 4). Thedermal thickness of skin explants injected with different concentrationsof E1 or E4 in combination with TGF-β was examined. As shown in FIG. 5,unlike E1, E4 at concentrations of 5-20 μg/ml clearly amelioratedTGF-β-induced skin fibrosis, indicating that the C-terminus ofendostatin can suppress fibrosis (see FIG. 17).

Example 6 Endostatin Peptides Reduce TGF-β-Induced Fibrosis In Vivo inMouse Skin

The anti-fibrotic effect of endostatin peptides was further assessed invivo. rE and endostatin peptides in combination with TGF-β were injectedin the skin of mice. One week post-injection, mice appeared healthy andshowed no signs of distress. As shown in FIG. 6, human TGF-β stronglyincreased dermal thickness in mouse skin (P=0.004). Peptides E3 and E4from the carboxy terminus of human endostatin peptide prevented dermalfibrosis induced by TGF-β (P=0.01, 0.007, respectively). In addition, E2significantly reduced dermal thickness (P=0.03). E1, a peptidecorresponding to the amino terminus of endostatin did not alterTGF-β-induced dermal fibrosis. These results confirmed those obtained inour human skin model and emphasize the importance of the C-terminaldomain of endostatin in preventing TGF-β-induced fibrosis in vivo and exvivo.

Example 7 The C-Terminal Peptide of Endostatin has ModestAnti-Angiogenic Activity

The anti-angiogenic effect of endostatin has been attributed to itsamino terminal domain (Tjin Tham Sjin et al., Cancer Res 2005;65:3656-63). To evaluate the anti-angiogenic capacity of the carboxyterminal regions of endostatin, the effect of E4 on in vitro tubularformation was examined using Matrigel. As shown in FIG. 7, the capacityof rE to inhibit tubular structure formation by HUVECs was significant,confirming previous reports. On the other hand, the ability of E4 tosuppress angiogenesis was modest, suggesting that the region ofendostatin corresponding to E4 does not significantly contribute to itsanti-angiogenic activity.

Thus, E4, a peptide corresponding to the carboxy terminal region ofendostatin, ameliorates TGF-β-induced fibrosis and even reverses it. E4suppressed TGF-β-induced ECM production and downregulated α-SMA levelsin primary lung and skin fibroblasts. In vivo and ex vivo analysesrevealed that E4 impedes the increase of skin dermal thickness triggeredby TGF-β. Furthermore, the anti-angiogeneic capacity of E4 was lowcompared to that of rE. Taken together, the findings suggest that thedomains of endostatin responsible for its anti-fibrotic andanti-angiogenic capacity are distinct. Other endostatin peptides (forexample, E2 and E3) are shown to have anti-fibrotic activity.

The anti-angiogenic activity of endostatin has been the focus ofnumerous investigations directed at the development of anti-tumortherapy. Recently, elevated serum and BALF levels of endostatin infibrotic disorders such as idiopathic pulmonary fibrosis (IPF) andsystemic sclerosis (SSc) were reported. Endostatin levels wererelatively increased in IPF patients with severe respiratory dysfunctionand in SSc patients with pulmonary fibrosis, severe skin fibrosis, andwith cutaneous scars, compared to patients without those clinicalmanifestations (Sumi J Clin Lab Anal 2005; 19:146-9; Richter et al.,Thorax 2009; 64:156-61). In addition, collagen XVIII expression wasincreased in cultured dermal fibroblasts of SSc patients (Tan et al.,Arthritis Rheum 2005; 52:865-76) and in whole lung extracts of patientswith IPF (Yang et al., Am J Respir Crit Care Med 2007; 175:45-54). Inthis regard, since endostatin is a proteolytic product of collagen XVIIIcleaved by several proteases including MMPs and cathepsin L (Wen et al.,Cancer Res 1999; 59:6052-6; Felbor, EMBO J 2000; 19:1187-94), and sinceMMPs are also upregulated in SSc and IPF (Richter et. al., Thorax 2009;64:156-61, Toubi et al., Clin Exp Rheumatol 2002; 20:221-4), theobservations that cleaved endostatin levels are elevated in thosepatients is plausible. However, it is unclear how endostatin may beinvolved in the pathogenesis of fibrosis.

Without being bound by theory, increased endostatin in fibrotic tissuesmay constitute a negative feedback regulatory loop which, althoughunsuccessful, is directed at halting the progression of fibrosis. Sinceendostatin was originally identified in aberrant “angiogenic”endothelial cancer cells as a product that likely controls/inhibits its“angiogenic” capacity (O'Reilly et al., Cell 1997; 88:277-85), it isplausible that endostatin in fibrosis serves a similar regulatoryfunction.

Bloch W et al reported reduced connective tissue but normal vesseldensity in recombinant endostatin-treated mouse skin using a woundhealing model (Bloch et al., FASEB J 2000; 14:2373-6). Furthermore, apeptide from the N-terminal region of endostatin prevented theprogression of peritoneal sclerosis in a mouse model (Tanabe et al.,Kidney Int 2007; 71:227-38); the peptide under investigationcorresponded to the N-terminus of endostatin encompassing amino acids1-27.

In contrast, the C-terminal region of endostatin, but not theN-terminus, is shown herein to be responsible for its anti-fibroticeffects. In fact, the peptide corresponding to the N-terminal domain ofendostatin contributed to the fibrotic phenotype in some of the assays.Studies directed at defining the specific amino acid sequenceresponsible for endostatin's anti-angiogenic capacity (Richter et al.,Thorax 2009; 64:156-61; Cattaneo et al., Exp Cell Res 2003; 283:230-6;Xu et al., Curr Protein Pept Sci 2008; 9:275-83) have shown that theentire angio-suppressive activity of endostatin was located in a27-amino-acid peptide in the N-terminal domain (Richter et al., Thorax2009; 64:156-61). Thus, the functional domain of endostatin thatmediates its anti-fibrotic activity is different from that responsiblefor its anti-angiogenic capacity, implying different mechanisms forinhibiting angiogenesis and fibrosis. The anti-fibrotic C-terminalendostatin polypeptides disclosed herein are therefore capable ofselectively inhibiting fribrosis without inhibiting angiogenesis. TheC-terminal endostatin polypeptides can be used to more specifically andselectively target unwanted fibrosis without interfering withangiogenesis that may impact a desired therapeutic outcome.

The C-terminal endostatin polypeptide also reduces α-SMA expression inTGF-β-treated fibroblasts. In addition, the matrix reducing effects ofE4 on normal fibroblasts was modest compared to that in fibroticfibroblasts. This suggests that the therapeutic effect of endostatinC-terminal peptide in fibrosis could be due, in part, to hindrance offibroblast activation by TGF-β and other fibrosis promoting growthfactors.

In 2005, ENDSTAR®, a recombinant human endostatin purified from E. colicontaining an additional nine-amino acid sequence produced as ahis-tagged protein was approved for the treatment of non-small-cell lungcancer in China (Sun et al., J Clin Oncol 2005 (ASCO Annual meetingproceedings); 23:7138). Despite its effectiveness, the treatment hadseveral disadvantages including a requirement for high doses, theprotein's short half-life, poor stability and easy inactivation (see,for example, Crystal, Nat Biotechnol 1999; 17:336-7; Hu et al., ActaPharmacol Sin 2008; 29:1357-69). The small synthetic peptides disclosedherein could overcome these obstacles. E4 significantly inhibitedfibrosis compared to rE and even E3 in vitro, in vivo, and ex vivo. Inaddition, E4 had minimal anti-angiogenic activity compared to rE,confirming that the anti-angiogenic activity of endostatin resides inits N-terminal domain. The only difference between E3 and E4 was thepresence of an amide-bond in the C-terminus of E4. Without being boundby theory, this amide renders the peptide more resistant to carboxydegradation by carboxypeptidases or other degrading molecules, thusstabilizing the peptide and likely maintaining its biological activity(Yang et. al. Am J Respir Crit Care Med 2007; 175:45-54).

Unfortunately, there are no effective therapies for organ fibrosis. TheC-terminal domain of endostatin, corresponding to amino acid sequence133-180 with amide-bond formation, suppressed ECM production by primaryskin and lung fibroblasts and ameliorated dermal fibrosis induced byTGF-β in vivo and ex vivo in human skin. The findings presented hereindemonstrate that E4 could be used for the treatment of fibroticdisorders, including IPF, SSc, morphea, as well as Graft-versus-hostdisease, keloid and hypertrophic scar, and other organ fibrosis such assubepithelial fibrosis in asthma.

Example 8 Confirmation of the Efficacy of E4

E4, a peptide representing the carboxy terminus of human endostatin, canattenuate fibrosis triggered by multiple fibrogenic factors. Theanti-fibrotic effects of E4 can be detected whether administeredconcomitantly with or following the fibrogenic trigger. The efficacy ofE4 was confirmed in four pre-clinical models of fibrosis: a)bleomycin-induced dermal fibrosis in vivo in mouse skin, b) TGF-βinduced dermal fibrosis in mouse skin, and c) bleomycin-inducedpulmonary fibrosis. E4 peptide or a control peptide (E1; representingthe amino terminal region of endostatin) were administered at the sametime as TGF-β or bleomycin or 3-4 days following TGF-(3 or bleomycin.Mice were sacrificed one and two weeks after TGF-(3-initiation of dermalfibrosis, and two and three weeks after bleomycin-induced pulmonaryfibrosis. Two different modes of administration of the E4 peptide werealso tested. It was confirmed that intraperitoneal and intratrachealadministration was effective. The amount of E4 that was administered was10 μg/ml in a total volume of 100 μl for skin and IP injections and 50μl for IT administration.

For these studies, fibrosis was assessed by measurement of dermalthickness on H&E skin sections (skin), assessment of collagen levels byMasson Trichrome staining (skin and lung), and measurement of collagenlevels by Sircol assay (lung). Furthermore, to confirm the mechanism bywhich E4 exerts its anti-fibrotic effects, the production ofextra-cellular matrix (ECM) components, the levels of enzymes thatpromote matrix stabilization and thus accumulation and levels of thosethat degrade ECM components, and levels of transcription factorsdownstream of the pro-fibrotic triggers were evaluated. Results wereassessed using the unpaired t test and the 3-way ANOVA (for the IDIdata).

Results

E4 caused a significant attenuation of bleomycin induced dermal fibrosiseven with a single administration of E4 (FIG. 8). E4 caused asignificant decrease of TGFbeta induced dermal fibrosis on day 7. ThusE4 prevents (FIG. 8) and reverses (FIG. 9) dermal fibrosis triggered byTGFbeta.

E4 administered concomitantly with bleomycin or three days followingbleomycin caused a marked reduction in fibrosis and Masson Trichromestaining (see FIG. 9 and FIG. 10). E4 peptide given three days afterbleomycin significantly reduced collagen levels in mouse lungs (FIG. 10,panel B).

E4 caused a statistically significant reduction in both TGFβ andbleomycin induced skin (FIG. 8) and lung fibrosis (FIG. 10) regardlessof the mode of administration. Intraperitoneal and intratrachealadministration of E4 were both effective in blocking dermal andpulmonary fibrosis. For example, E4 caused a significant attenuation ofbleomycin induced lung fibrosis on day 21 whether administeredintraperitoneally or intratracheally (FIG. 11). Thus E4 is effective atreducing fibrosis irrespective of the administration mode.

The results also evidenced that E4 exerts its anti-fibrotic effects viamultiple pathways. E4 reduces levels of lysyl oxidase (LOX), and enzymeresponsible for the cross-linking of collagen, elastin, and otherextracellular matrix (ECM) molecules and thus the stabilization of theECM. E4 can make collagen less stable and more susceptible toproteolytic degradation. FIG. 12 shows lung sections of mice treatedwith bleomycin with or without E4.

E4-mediated reduction of LOX was detected also was detected in vitro.Normal lung fibroblasts in passage 4 were treated with vehicle, E4,TGFβ, or TGF-β followed 30 minutes later by E4 (FIG. 13). Mediaconditioned by the fibroblasts were analyzed using western blot analysisafter 48 hrs. Treatment with E4 significantly reduced the level of LOX.Similar results were obtained when LOX mRNA levels were examined byreal-time PCR

E4 also promotes the degradation of ECM components via induction andactivation of matrix metalloprotease (MMP-2), an enzyme that degradesseveral ECM molecules including fibronectin and native and denaturedcollagens (FIG. 14). In addition, E4 increases levels of inhibitor ofdifferentiation (ID)-1, a transcription factor that inhibits TGF-βeffects (see FIG. 15). It was determined in a Western bloat analysisthat E4 reduces the levels of the master switch transcription factor,Egr-1 (see FIG. 16) in primary human lung fibroblasts, treated andharvested after 24 hours. The reduction of Egr-1 levels parallels areduction in collagen, SMA and fibronectin. Egr-1 is known to mediatethe effects of several fibrotic agents (including TGF-β and bleomycin).

Thus, E4 exerted significant anti-fibrotic effects. This peptidesignificantly attenuates the fibrogenic effects of TGFbeta and bleomycinwhether administered simultaneously with these fibrotic triggers or afew days following the initiation of fibrosis, suggesting that E4, andother C-terminal endostatin polypeptides is also effective at reversingestablished fibrosis. The anti-fibrotic effects of E4 were noted whetherit was administered intratracheally or intraperitoneally to mice inwhich pulmonary fibrosis was induced by bleomycin and dermal fibrosiswas induced by TGFbeta. Furthermore, E4 exerted its anti-fibroticeffects via multiple pathways that include destabilization of ECMthrough reduction of LOX and thus decrease of ECM crosslinking,induction of ECM degradation via activation of MMP-2, suppression ofEgr-1 levels, and induction of the TGFβ, thereby inhibitingtranscription factor ID-1.

Thus, several in vitro assays and four in vivo and ex vivo pre-clinicalmodels of fibrosis suggest that C-terminal endostatin polypeptides, asexemplified by E4, are an effective anti-fibrotic peptide that can blockand reverse fibrosis in two organs, lung and skin. These anti-fibroticeffects as well as the lack of anti-angiogenic effects characteristic ofendostatin render E4 an attractive therapeutic peptide for organfibrosis.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An isolated polypeptide, comprising at least 40 consecutive aminoacids of the amino acid sequence set forth as amino acids 133-180 of SEQID NO: 2, with at most 5 amino acid substitutions, wherein thepolypeptide has anti-fibrotic activity and wherein the polypeptide doesnot comprise amino acids 1-92 or SEQ ID NO:
 2. 2. The isolatedpolypeptide of claim 1, comprising at least 40 consecutive amino acidsof the amino acid sequence set forth as amino acids 133-180 of SEQ IDNO:
 2. 3. (canceled)
 4. The isolated polypeptide of claim 1, consistingessentially of (a) the amino acid sequence set forth as amino acids133-180 of SEQ ID NO: 2; or (b) the amino acid sequence set forth asamino acids 133-180 of SEQ ID NO: 2 with at most 5 amino acidsubstitutions; wherein the carboxy terminus of the polypeptide isoptionally amidated.
 5. The isolated polypeptide of claim 1, consistingof (1) 40 to 48 consecutive amino acids of the amino acid sequence setforth as amino acids 133-180 of SEQ ID NO: 2; (2) 40 to 48 consecutiveamino acids of the amino acid sequence set forth as amino acids 133-180of SEQ ID NO: 2, with at most 5 amino acid substitutions, (3) the aminoacid sequence set forth as amino acids 133-180 of SEQ ID NO: 2; (4) theamino acid sequence set forth as amino acids 133-180 of SEQ ID NO: 2with at most 5 amino acid substitutions; (5) any of (1)-(4) wherein thecarboxy terminus of the polypeptides is amidated.
 6. The isolatedpolypeptide of claim 1, comprising (1) at least 40 consecutive aminoacids of the amino acid sequence set forth as amino acids 133-180 of SEQID NO: 13, wherein the polypeptides has anti-fibrotic activity; (2) atleast 40 consecutive amino acids of the amino acid sequence set forth asamino acids 133-180 of SEQ ID NO: 13, with at most 5 amino acidsubstitutions, (3) the amino acid sequence set forth as amino acids133-180 of SEQ ID NO: 13; or (4) the amino acid sequence set forth asamino acids 133-180 of SEQ ID NO: 13 with at most 5 amino acidsubstitutions; wherein the carboxy terminus of the polypeptide isoptionally amidated.
 7. The isolated polypeptide of claim 1, wherein thecarboxy terminus of the polypeptide is amidated.
 8. The isolatedpolypeptide of claim 7, wherein the polypeptide comprises amino acids133-180 of SEQ ID NO: 2 or amino acids 133-180 of SEQ ID NO: 13, andwherein the carboxy terminus of the polypeptide is amidated.
 9. Theisolated polypeptide of claim 1, wherein the polypeptide consists ofamino acids 133-180 of SEQ ID NO: 2 or amino acids 133-180 or SEQ ID NO:13 wherein the carboxy terminus of the polypeptide is amidated.
 10. Anisolated polynucleotide encoding the polypeptide of claim
 1. 11. Theisolated polynucleotide of claim 10, operably linked to a promoter. 12.An expression vector comprising the isolated polypeptide of claim 11.13. An isolated host cell transformed with the expression vector ofclaim
 12. 14. The isolated host cell of claim 13, wherein the host cellis a mammalian cell or an insect cell.
 15. A pharmaceutical compositioncomprising the isolated polypeptide of claim 1 or an isolatedpolynucleotide encoding the polypeptide and a pharmaceuticallyacceptable carrier.
 16. A method of treating a subject with fibrosis,comprising administering to the subject a therapeutically effectiveamount of the polypeptide of claim 1, thereby treating the subject withfibrosis.
 17. The method of claim 16, wherein the subject has a fibrosisof the skin.
 18. The method of claim 17, wherein the subject hasscleroderma.
 19. The method of claim 16, wherein the subject has akeloid or hypertrophic scar.
 20. The method of claim 16, wherein thesubject has idiopathic pulmonary fibrosis, morphea, Graft-Versus-HostDisease, or subepithelial fibrosis.
 21. The method of claim 16, furthercomprising administering to the subject a therapeutically effectiveamount of another therapeutic agent. 22-24. (canceled)
 25. A method fordecreasing lysyl oxidase production by a cell, comprising contacting thecell with an effective amount of claim 1, thereby decreasing theproduction of lysyl oxidase by the cell.
 26. The method of claim 25,wherein the cell is in vitro.
 27. (canceled)
 28. A method for increasingthe production of matrix metalloprotease by a cell, comprisingcontacting the cell with an effective amount of the polypeptide of anyof claims 1-9, thereby increasing the production of matrixmetalloprotease by the cell.
 29. The method of claim 28, wherein thecell is in vitro. 30-43. (canceled)